TTK in diagnosis and as a therapeutic target in cancer

ABSTRACT

The present invention provides methods for identification of cancerous cells by detection of expression levels of TTK, as well as diagnostic, prognostic and therapeutic methods that take advantage of the differential expression of these genes in mammalian cancer. Such methods can be useful in determining the ability of a subject to respond to a particular therapy, e.g., as the basis of rational therapy. In addition, the invention provides assays for identifying pharmaceuticals that modulate activity of these genes in cancers in which these genes are involved, as well as methods of inhibiting tumor growth by inhibiting activity of TTK.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisionalapplication serial No. 60/289,813, filed Feb. 21, 2001, whichapplication is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The field of the present invention relates to disease diagnosisand treatment of cancer and identification of anti-cancer agents.

BACKGROUND OF THE INVENTION

[0003] Mitotic checkpoint genes have become widely studied for theirroles in development as well as for their potential role in disease suchas cancer. The mitotic checkpoint involves a number of differentmechanisms to ensure proper cellular division. For example, the spindleassembly checkpoint modulates the timing of anaphase initiation inresponse to the improper alignment of chromosomes at the metaphaseplate. If defects are detected, a signal is transduced to halt furtherprogression of the cell cycle until correct bipolar attachment to thespindle is achieved. Initially identified in budding yeast, severalmammalian spindle checkpoint-associated proteins have recently beenidentified and partially characterized. These proteins associate withall active human centromeres, including neocentromeres, in the earlystages of mitosis prior to the commencement of anaphase. The proteinsassociated with the checkpoint protein complex (BUB1, BUBR1, BUB3,MAD2), the anaphase-promoting complex (Tsg24, p55CDC), and otherproteins associated with mitotic checkpoint control (ERK1, 3F3/2epitope, hZW10), were found to specifically associate with only theactive centromere, suggesting that only active centromeres participatein the spindle checkpoint. Saffery R et al., Hum Genet. 107:376-84(2000).

[0004] Tyrosine threonine kinase (TTK), a protein kinase, phosphorylatesserine, threonine, and tyrosine hydroxyamino acids (Mills et al,. Biol.Chem. 267:16000-6 (1992)). The kinases most closely related to TTKinclude SPK1 serine, threonine, and tyrosine kinase, the Pim1, PBS2, andCDC2 serine/threonine kinases, and the TIK kinase (Mills et al. J. Biol.Chem. 267:16000 -6 (1992)). The nucleotide and amino acid sequences ofhuman TTK are provided at, for example, GenBank Accession No. M86699.Expression of TTK is markedly reduced or absent in resting cells and intissues with a low proliferative index (Hogg et al. Oncogene 9:89-96(1994)). TTK mRNA is expressed in human testis, thymus, bone marrow, andother tissues that contain a large number of proliferating cells and isnot detected in tissues that contain few or no dividing cells. TTKexpression was detected in several rapidly proliferating cells lines,including various cancer cell lines. TTK expression was also detectedand in samples tissue samples from two patients with malignant ovariancancer (Mills et al., ibid.; Schmandt et al. J. Immunol. 152:96-105(1994)). TTK expression is correlated with cell proliferation, and playsa role in cell cycle control (Hogg et al., ibid.). Very low levels ofTTK mRNA and protein are present in starved cells. When cells areinduced to enter the cell cycle, levels of TTK mRNA, protein, and kinaseactivity increase at the G1/S phase of the cell cycle and peak in G2/M.TTK mRNA levels, as well as kinase activity, drop sharply in early G1,whereas protein levels are largely maintained. TTK is a human homologueof the S. cerevesiae kinase mps1 and the S. pombe protein mph1, both ofwhich are involved in cell cycle spindle assembly checkpoint, thusindicating that TTK is a spindle checkpoint gene (see, e.g., Cahill etal. Genomics 58:181-7 (1999).

[0005] Although mitotic checkpoint impairment has been detected in humancancers (e.g., such impairment is present in about 40% of human lungcancer cell lines) mutations in the MAD mitotic checkpoint genes and theBUB gene family are infrequent. Haruki N et al., Cancer Lett.162:201-205 (2001); Mimori K et al., Oncol Rep. 8:39-42 (2001); Cahillet al., ibid.). There is thus a need for identification of mitoticcheckpoint genes that have a role in human cancers, as they can serve asinformative diagnostic and/or prognostic indicators, and therapeutictargets.

SUMMARY OF THE INVENTION

[0006] The present invention provides methods for identification ofcancerous cells by detection of expression levels of TTK, as well asdiagnostic, prognostic and therapeutic methods that take advantage ofthe differential expression of these genes in mammalian cancer. Suchmethods can be useful in determining the ability of a subject to respondto a particular therapy, e.g., as the basis of rational therapy. Inaddition, the invention provides assays for identifying pharmaceuticalsthat modulate activity of these genes in cancers in which these genesare involved, as well as methods of inhibiting tumor growth byinhibiting activity of TTK.

[0007] In a first embodiment, the present invention provides a methodfor identifying TTK levels in a sample of a subject suspected of havingcancer (e.g., a lung, colon, prostrate or breast tissue biopsy)comprising quantifying the level of TTK in the sample. Theidentification of increased levels of TTK in the sample provides anindication of impairment of the cell cycle checkpoint in the sampledcells.

[0008] In another embodiment, the invention provides a method fordetermining the characteristics of a malignant or pre-malignant growthcomprising determining (either qualitatively or quantitatively) thelevel of TTK in the cells of the growth, and comparing levels with knownlevels in various stages of cancer and/or normal tissue. For example, todetermine the characteristics of a particular subject's colon cancer, asample of the cancer may be removed, the levels of TTK in the cancerdetermined, and the levels compared to normal tissue and/or levels invarious stage colon cancers derived from the same cell type. The levelsof TTK identified in the sample can thus be indicative of variouscharacteristics of the malignant or pre-malignant growth, as determinedby the characteristics of known tissue and cancers. The TTK levels canbe compared directly to the levels in other single samples, or may becompared to a standard that is derived from the data of multiplesamples.

[0009] In another embodiment, the TTK levels of a sample can be used asone index for determining the appropriate therapeutic intervention for asubject with a malignant or pre-malignant growth. Highly increasedlevels of TTK, for example, can be indicative of the need for moreaggressive therapy, as it is indicative of a later stage cancer.Alternatively, the level of TTK expression may be indicative of theresponsiveness of a subject to a particular pharmaceutical, and inparticular to a therapeutic intervention that affects the cancer via themitotic checkpoint.

[0010] In another embodiment, the invention features a method foridentifying agents for inhibiting growth of a tumor, particular by abreast or colon tumor, by contacting a cell expressing TTK with acandidate agent, and assessing the effect of the agent upon TTKactivity.

[0011] Accordingly, in one aspect the invention features a method ofdiagnosing cancer in a subject, the method comprising detection of TTKpolynucleotide or polypeptide in a test sample obtained from a subjectso as to determine a level of expression of the gene product; andcomparing the level of expression of the TTK in the test sample to alevel of expression in a normal cell corresponding to the same tissue;wherein detection of an expression level of TTK in the test sample thatis significantly increased from the level of expression in a normal cellindicates that the test cell is cancerous. In specific embodiments, thecancer is other than ovarian cancer, with colon cancer and breast cancerbeing of particular interest.

[0012] In another aspect, the invention features a method fordetermining the prognosis of a cancerous disease in a subject, themethod comprising detecting expression of TTK in a test cell from thesubject; and comparing a level of expression of TTK in the test cellwith a level of TTK expression in a control cell; wherein the level ofexpression of TTK in the test cell relative to the level of expressionin the control cell is indicative of the prognosis of the cancerousdisease. For example, where the control cell is a normal cell, anelevated level of TTK expression in the test cell relative to the normalcell is indicative of the continued presence of cancerous cells in thesubject and thus a relatively poorer prognosis than where the level ofTTK expression in the test cell is at a level comparable to that foundin an normal (non-cancer) cell. In specific embodiments, progress of acancer other than ovarian cancer is of particular interest, especiallycolon and breast cancer.

[0013] In another aspect, the invention features a method for inhibitinggrowth of a cancerous cell comprising introducing into a cell anantisense polynucleotide for inhibition of TTK expression, whereininhibition of TTK expression inhibits replication of the cancerous cell.

[0014] In still another aspect, the invention features a method forassessing the tumor burden of a subject, the method comprising detectinga level of TTK expression in a test sample from a subject, the testsample suspected of comprising increased TTK expression; whereindetection of the level of TTK expression in the test sample isindicative of the tumor burden in the subject, with an increased levelof TTK expression in the test sample relative to a control non-cancercell indicates the presence of a tumor in the subject.

[0015] In yet another aspect, the invention features a method ofidentifying an agent having anti-TTK activity, the method comprisingcontacting a cancerous cell displaying elevated expression of TTK with acandidate agent; and determining the effect of the candidate agent onTTK activity; wherein a decrease in TTK activity indicates that theagent has anti-TTK activity. In specific embodiments, TTK activity isdetected by detecting TTK expression or by detecting a biologicalactivity of TTK

[0016] In yet another aspect, the invention features an assay foridentifying a candidate agent that inhibits growth of a cancerous cell,comprising contacting a cell expressing TTK polypeptide with a candidateagent; and detecting activity of the TTK polypeptide, comparing theactivity of the TTK polypeptide in the cell in the presence of thecandidate agent to activity of a TTK polypeptide in a cell in theabsence of the candidate agent; wherein reduction of TTK activity in thepresence of the candidate agent relative to TTK activity in the absenceof the candidate agent indicates that the candidate agent reduces TTKactivity and inhibits growth of a cancerous cell.

[0017] A primary object of the invention is to exploit TTK as atherapeutic target, e.g. by identifying candidate agents that modulate,usually that decrease, TTK activity in a target cell in order to, forexample, inhibit cell growth.

[0018] An object of the present invention is to inhibit tumor growth byinhibition of activity of a mitotic checkpoint gene product,particularly though inhibition of TTK activity in the target tumor cell.

[0019] Another object of the invention is to facilitate rational cancertherapy. For example, where the cancer in the subject is associated withincreased TTK activity levels, a therapeutic agent is selectedaccordingly so as to facilitate reduction of TTK activity levels.

[0020] Another object of the present invention is to design clinicaltrials based on levels of TTK expression in a cancer, and moreparticularly to design clinical trials based on TTK expression incombination with other patient attributes.

[0021] Yet another object of the invention is to identify theassociation of TTK expression and intervention attributes that yieldefficacious changes in selected disease progression measures.

[0022] An advantage of the invention is the ability to project diseaseprogression based on expression of TTK in a malignant or pre-malignantgrowth.

[0023] Another advantage of the present invention is that it allows amore systematic approach for intervention of a cancerous disease basedupon objective indicia.

[0024] These and other objects, advantages, and features of theinvention will become apparent to those persons skilled in the art uponreading the details of the methods as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a bar graph illustrating expression of TTK in variousnormal tissue types as detected by PCR.

[0026]FIG. 2 is a bar graph illustrating expression of TTK in varioustumor cell lines as detected by PCR.

[0027] FIGS. 3-6 are graphs illustrating expression profiles for IGF2,MAPKAPK2, TTK, and MARCKS in patients with colorectal carcinoma.

[0028]FIGS. 7 and 8 are graphs illustrating growth suppression ofMDA-MB-231 cells following antisense suppression of TTK expression.

[0029]FIG. 9 is a graph illustrating growth suppression of SW620 cellsfollowing antisense suppression of TTK expression.

[0030]FIG. 10 is a graph illustrating suppression of colony formation ofSW620 cells in soft agar following antisense suppression of TTKexpression.

[0031]FIG. 11 is a graph illustrating that antisense suppression of TTKhas no detectable effect on normal immortal fibroblasts.

[0032]FIG. 12 is a bar graph illustrating induction of cell death upondepletion of TTK from SW620 cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0033] Before the present invention is described, it is to be understoodthat this invention is not limited to particular methodologiesdescribed, and as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

[0034] Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

[0035] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

[0036] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a cell” includes a plurality of such cells and reference to “the agent”includes reference to one or more agents and equivalents thereof knownto those skilled in the art, and so forth.

[0037] The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

[0038] The terms “polynucleotide” and “nucleic acid”, usedinterchangeably herein, refer to a polymeric forms of nucleotides of anylength, either ribonucleotides or deoxynucleotides. Thus, these termsinclude, but are not limited to, single-, double-, or multi-stranded DNAor RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprisingpurine and pyrimidine bases or other natural, chemically orbiochemically modified, non-natural, or derivatized nucleotide bases.These terms further include, but are not limited to, mRNA or cDNA thatcomprise intronic sequences (see, e.g., Niwa et al. (1999) Cell99(7):691-702). The backbone of the polynucleotide can comprise sugarsand phosphate groups (as may typically be found in RNA or DNA), ormodified or substituted sugar or phosphate groups. Alternatively, thebackbone of the polynucleotide can comprise a polymer of syntheticsubunits such as phosphoramidites and thus can be anoligodeoxynucleoside phosphoramidate or a mixedphosphoramidate-phosphodiester oligomer. Peyrottes et al. (1996) Nucl.Acids Res. 24:1841-1848; Chaturvedi et al. (1996) Nucl. Acids Res.24:2318-2323. A polynucleotide may comprise modified nucleotides, suchas methylated nucleotides and nucleotide analogs, uracyl, other sugars,and linking groups such as fluororibose and thioate, and nucleotidebranches. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.Other types of modifications included in this definition are caps,substitution of one or more of the naturally occurring nucleotides withan analog, and introduction of means for attaching the polynucleotide toproteins, metal ions, labeling components, other polynucleotides, or asolid support.

[0039] The terms “polypeptide” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;and the like.

[0040] As used herein “TTK polynucleotide” and “TTK polypeptide”encompass polynucleotides and polypeptides having sequence similarity orsequence identity to the human TTK (having GenBank accession numberM86699; SEQ ID NO:13 and 14), or the S. cerevesiae kinase mps1 gene andgene products (SEQ ID NO:29 and 30), the S. pombe protein mph1 gene andgene products (SEQ ID NO:31 and 32), and other genes and gene productsrelated to TTK, such as SPK1 (SEQ ID NO:15 and 16), Pim1 (SEQ ID NO:17and 18), PBS2 (SEQ ID NO:19 and 20), CDC2 (SEQ ID NO:21 and 22), and TIK(SEQ ID NO:23 and 24) of at least about 65%, preferably at least about80%, more preferably at least about 85%, and can be about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Sequence similarity andsequence identity are calculated based on a reference sequence, whichmay be a subset of a larger sequence, such as a conserved motif, codingregion, flanking region, etc. A reference sequence will usually be atleast about 18 nt long, more usually at least about 30 nt long, and mayextend to the complete sequence that is being compared. In general,percent sequence identity is calculated by counting the number ofresidue matches (e.g., nucleotide residue or amino acid residue) betweenthe query and test sequence and dividing total number of matches by thenumber of residues of the individual sequences found in the region ofstrongest alignment. Thus, where 10 residues of an 11 residue querysequence matches a test sequence, the percent identity above would be 10divided by 11, or approximately, 90.9%. Algorithms for computer-basedsequence analysis are known in the art, such as BLAST (see, e.g.,Altschul et al., J. Mol. Biol., 215:403-10 (1990)), particularly theSmith-Waterman homology search algorithm as implemented in MPSRCHprogram (Oxford Molecular). For the purposes of this invention, apreferred method of calculating percent identity is the Smith-Watermanalgorithm, using the following. Global DNA sequence identity must begreater than 65% as determined by the Smith-Waterman homology searchalgorithm as implemented in MPSRCH program (Oxford Molecular) using anaffine gap search with the following search parameters: gap openpenalty, 12; and gap extension penalty, 1. The human TTK cDNA isrepresented by the polynucleotide sequence of SEQ ID NO:13 and the humanTTK polypeptide is represented by the sequence of SEQ ID NO:14.

[0041] “Antisense polynucleotide” or “antisense oligonucleotide” areused interchangeably herein to mean an unmodified or modified nucleicacid having a nucleotide sequence complementary to a givenpolynucleotide sequence (e.g., a polynucleotide sequence encoding TTK)including polynucleotide sequences associated with the transcription ortranslation of the given polynucleotide sequence (e.g., a promoter of apolynucleotide encoding TTK), where the antisense polynucleotide iscapable of hybridizing to a TTK-encoding polynucleotide sequence. Ofparticular interest are antisense polynucleotides capable of inhibitingtranscription and/or translation of a TTK-encoding polynucleotide eitherin vitro or in vivo.

[0042] The term “cDNA” as used herein is intended to include all nucleicacids that share the arrangement of sequence elements found in nativemature mRNA species, where sequence elements are exons (e.g., sequencesencoding open reading frames of the encoded polypeptide) and 3′ and 5′non-coding regions. Normally mRNA species have contiguous exons, withthe intervening introns removed by nuclear RNA splicing to create acontinuous open reading frame encoding TTK.

[0043] A “variant” as used in the context of a “variant polypeptide”refers to an amino acid sequence that is altered by one or more aminoacids relative to a reference amino acid sequence. The variant can have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant can have “nonconservative” changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations can also include amino acid deletions or insertions, or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted, or deleted without abolishing biological orimmunological activity can be found using computer programs well knownin the art, for example, DNAStar software.

[0044] A “deletion” is defined as a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent as compared to reference amino acidsequence or nucleotide sequence. Deletions can be of any length, but arepreferably approximately 50, 20, 15, 10, 5 or 3 amino acids ornucleotides in length.

[0045] An “insertion” or “addition” is that change in an amino acid ornucleotide sequence which has resulted in the addition of one or moreamino acid or nucleotide residues, respectively, as compared to areference amino acid sequence or nucleotide sequence. Insertions oradditions can be of any length, but are preferably approximately 50, 20,15, 10, 5 or 3 amino acids or nucleotides in length.

[0046] A “substitution” results from the replacement of one or moreamino acids or nucleotides by different amino acids or nucleotides,respectively, as compared to a reference amino acid sequence ornucleotide sequence. Substitutions can be of any length, but arepreferably approximately 50, 20, 15, 10, 5 or 3 amino acids ornucleotides in length.

[0047] The terms “single nucleotide polymorphism” and “SNP” refer topolymorphisms of a single base change relative to a reference sequence.

[0048] The term “biologically active” refers to gene product, usually apolypeptide, having structural, regulatory, or biochemical functions ofa naturally occurring gene product, e.g., protein. “Immunologicallyactive” defines the capability of the natural, recombinant, or syntheticpolypeptide, or any oligopeptide thereof, to elicit a specific immuneresponse in appropriate animals or cells and to bind with specificantibodies.

[0049] The term “derivative” as used herein refers to the chemicalmodification of a nucleic acid or amino acid sequence relative to areference nucleic acid or amino acid sequence. Illustrative of suchmodifications would be replacement of hydrogen by an alkyl, acyl, oramino group. A nucleic acid derivative generally encodes a polypeptidewhich retains essential biological characteristics of the polypeptideencoded by the reference nucleic acid (e.g., the “parent” molecule).

[0050] As used herein the term “isolated” is meant to describe acompound of interest (e.g., either a polynucleotide or a polypeptide)that is in an environment different from that in which the compoundnaturally occurs. “Isolated” is meant to include compounds that arewithin samples that are substantially enriched for the compound ofinterest and/or in which the compound of interest is partially orsubstantially purified.

[0051] As used herein, the term “substantially purified” refers to acompound (e.g., either a polynucleotide or a polypeptide) that isremoved from its natural environment and is at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which it is naturally associated.

[0052] “Stringency” typically occurs in a range from about Tm −5° C. (5°C. below the Tm of the probe or antibody) to about 20° C. to 25° C.below Tm. As will be understood by those of skill in the art, astringency hybridization can be used to identify or detect identicalpolynucleotide sequences or to identify or detect similar or relatedpolynucleotide sequences.

[0053] The term “hybridization” as used herein shall include “anyprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing” (Coombs, Dictionary of Biotechnology,Stockton Press, New York N.Y. (1994)). Amplification as carried out inthe polymerase chain reaction technologies is described in Dieffenbachet al., PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview N.Y. (1995).

[0054] The term “transformation” as used herein refers to a permanent ortransient genetic change, induced in a cell following incorporation ofnew DNA (i.e., DNA exogenous to the cell). Genetic change can beaccomplished either by incorporation of the new DNA into the genome ofthe host cell, or by transient or stable maintenance of the new DNA asan episomal element. Where the cell is a mammalian cell, a permanentgenetic change is generally achieved by introduction of the DNA into thegenome of the cell.

[0055] The term “construct” as used herein refers to a recombinantnucleic acid, generally recombinant DNA, that has been generated for thepurpose of the expression of a specific nucleotide sequence(s), or is tobe used in the construction of other recombinant nucleotide sequences.

[0056] As used herein, the term “differentially expressed” generallyrefers to a polynucleotide that is expressed at levels in a test cellthat differ significantly from levels in a reference cell, e.g., mRNA isfound at levels at least about 25%, at least about 50% to about 75%, atleast about 90% increased or decreased, generally at least about1.2-fold, at least about 1.5-fold, at least about 2-fold, at least about5-fold, at least about 10-fold, or at least about 50-fold or moreincreased or decreased in a cancerous cell when compared with a cell ofthe same type that is not cancerous. The comparison can be made betweentwo tissues, for example, if one is using in situ hybridization oranother assay method that allows some degree of discrimination amongcell types in the tissue. The comparison may also be made between cellsremoved from their tissue source. “Differential expression” refers toboth quantitative, as well as qualitative, differences in the genes'temporal and/or cellular expression patterns among, for example, normaland neoplastic tumor cells, and/or among tumor cells which haveundergone different tumor progression events.

[0057] The terms “correspond to” or “represents” as used in, forexample, the phrase “polynucleotide corresponds to a differentiallyexpressed gene” are used to refer to the relationship between a givenpolynucleotide and the gene from which the polynucleotide sequence isderived (e.g., a polynucleotide that is derived from a coding region ofthe gene, a splice variant of the gene, an exon, and the like) or towhich the polynucleotide hybridizes to under stringer conditions.

[0058] “Differentially expressed polynucleotide” as used herein refersto a nucleic acid molecule (RNA or DNA) comprising a sequence thatrepresents or corresponds to a differentially expressed gene, e.g., thedifferentially expressed polynucleotide comprises a sequence (e.g., anopen reading frame encoding a gene product; a non-coding sequence) thatuniquely identifies a differentially expressed gene so that detection ofthe differentially expressed polynucleotide in a sample is correlatedwith the presence of a differentially expressed gene in a sample.“Differentially expressed polynucleotides” is also meant to encompassfragments of the disclosed polynucleotides, e.g., fragments retainingbiological activity, as well as nucleic acids homologous, substantiallysimilar, or substantially identical (e.g., having about 90% sequenceidentity) to the disclosed polynucleotides.

[0059] “Diagnosis” as used herein generally includes determination of asubject's susceptibility to a disease or disorder, determination as towhether a subject is presently affected by a disease or disorder,prognosis of a subject affected by a disease or disorder (e.g.,identification of pre-metastatic or metastatic cancerous states, stagesof cancer, or responsiveness of cancer to therapy), and therametrics(e.g., monitoring a subject's condition to provide information as to theeffect or efficacy of therapy).

[0060] As used herein, the term “a polypeptide associated with cancer”(e.g., as in polypeptide associated with colon cancer) refers to apolypeptide that is present at relatively higher or lower levels in acancer cell relative to a normal cell of the same type.

[0061] The term “biological sample” encompasses a variety of sampletypes obtained from an organism and can be used in a diagnostic ormonitoring assay. The term encompasses blood and other liquid samples ofbiological origin, solid tissue samples, such as a biopsy specimen ortissue cultures or cells derived therefrom and the progeny thereof. Theterm encompasses samples that have been manipulated in any way aftertheir procurement, such as by treatment with reagents, solubilization,or enrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

[0062] The terms “treatment”, “treating”, “treat” and the like are usedherein to generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; orrelieving the disease symptom, i.e., causing regression of the diseaseor symptom. Thus “treatment of cancer” thus encompasses one or more ofinhibition of cellular proliferation, inhibition of metastasis, and thelike.

[0063] The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans. Othersubjects may include cattle, dogs, cats, guinea pigs, rabbits, rats,mice, horses, and so on.

[0064] The phrase “specific binding pair” as used herein comprises aspecific binding member and a binding partner which have a particularspecificity for each other and which bind to each other in preference toother molecules under stringent conditions. Examples of specific bindingpairs are antigens and antibodies, molecules and receptors andcomplementary nucleotide sequences. Other examples of binding pairs willbe apparent to one skilled in the art upon reading the presentdisclosure. Further, the term “specific binding pair” is also applicablewhere either or both of the specific binding member and the bindingpartner comprise a part of a larger molecule. In embodiments in whichthe specific binding pair are nucleic acid sequences, they arepreferably between 10 to 200 nucleotides long, more preferably greaterthan 15 to 100 nucleotides long.

[0065] By “antibody” is meant an immunoglobulin protein which is capableof binding an antigen. Antibody as used herein is meant to include theentire antibody as well as any antibody fragments (e.g., F(ab′)₂, Fab′,Fab, Fv) capable of binding the epitope, antigen, or antigenic fragmentof interest.

[0066] Antibodies of the invention are immunoreactive or immunospecificfor and therefore specifically and selectively bind to a protein ofinterest, e.g., human TTK protein. Antibodies which are immunoreactiveand immunospecific for human TTK are preferred. Antibodies for human TTKare preferably immunospecific—i.e., not substantially cross-reactivewith related materials, although they may recognize TTK homologs acrossspecies. The term “antibody” encompasses all types of antibodies (e.g.,monoclonal and polyclonal).

[0067] By “binds specifically” is meant high avidity and/or highaffinity binding of an antibody to a specific polypeptide, e.g., epitopeof a TTK protein. Antibody binding to its epitope on this specificpolypeptide is stronger than binding of the same antibody to any otherepitope, particularly those which may be present in molecules inassociation with, or in the same sample, as the specific polypeptide ofinterest. Antibodies which bind specifically to a polypeptide ofinterest may be capable of binding other polypeptides at a weak, yetdetectable, level (e.g., 10% or less of the binding shown to thepolypeptide of interest). Such weak binding, or background binding, isreadily discernible from the specific antibody binding to the compoundor polypeptide of interest, e.g., by use of appropriate controls.

[0068] The terms “cancer”, “neoplasm”, “tumor”, and the like are usedinterchangeably herein to refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation. Ingeneral, cells of interest for detection or treatment in the presentapplication include pre-malignant (e.g., benign hyperplasiac),malignant, metastatic, and non-metastatic cells.

[0069] “TTK activity” as used herein refers to activity of the TTKpolypeptide in phosphorylation of a recipient substrate.

[0070] “Modulation of TTK activity” as used herein refers to an increaseor decrease in TTK activity that can be a result of, for example,interaction of an agent with a TTK polypeptide (e.g., reversible orirreversible binding of an inhibitory agent so as to interfere with TTKpolypeptide interaction with a donor molecule or a recipient (acceptor)molecule in the phosphorylation activity of TTK), inhibition of TTKtranscription and/or translation (e.g., through antisense interactionwith the TTK gene or TTK transcript, through modulation of transcriptionfactors that facilitate TTK expression), and the like. Modulation of TTKactivity that results in a decrease of TTK activity is of particularinterest in the invention. In this context, TTK activity can bedecreased by an inhibitory agent at least 10%, 25%, 50%, 75%, 85%, 90%,up to 100% relative to TTK activity in the absence of an agent. TTKactivity can be assessed by assaying enzymatic activity, by assessingTTK polypeptide levels, or by assessing TTK transcription levels.Comparisons of TTK activity can also be accomplished by comparing TTKactivity assessed (either qualitatively or quantitatively) in a testsample to a standard TTK activity (e.g., a level of TTK activity in theabsence of an inhibitory agent or agonist, that is associated with anormal cell, a level of TTK activity of a cancerous cell of a selectedtissue type, and the like).

[0071] Overview

[0072] Human TTK is a mitotic checkpoint gene which encodes an 857 aminoacid protein that exhibits activity of a mixed specificity (tyr/thr)kinase. TTK is expressed in rapidly proliferating tissues such as testisand thymus. See, e.g., Mills G B et al., J Biol Chem. 267:16000-6(1992). The present invention is based upon the finding that TTK isdifferentially expressed in colon tumor cells relative to normal coloncells as detected by microarray analysis. Differential expression wasconfirmed in cell lines derived from various forms of cancer, indicatingthat the involvement of TTK in cancer as a more general mechanism. Inaddition, disruption of TTK function using antisense oligonucleotides to“knock-out” TTK message decreased proliferation, inhibited anchorageindependent growth, and induced apoptosis of cancer cell lines,including a metastatic breast cancer cell line (MDA-MB-213) and acolorectal carcinoma cell line (SW620). These data indicate that TTK canbe a therapeutic target for chemotherapy in cancers in which TTK isoverexpressed.

[0073] The identification of the association of TTK with cancer, and theconfirmation that inhibition of TTK activity (e.g., by reducing TTKexpression) serves as the basis for the materials and methods of theinvention, such as are disclosed and discussed herein, for use in, forexample, diagnosing cancer of a patient, particularly a cancer that issusceptible to treatment by decreasing activity of TTK. The inventionalso provides for planning and selection of appropriate therapeuticand/or prophylactic treatment, permitting streamlining of treatment bytargeting those most likely to benefit. The invention also provides fortreatment of a cancer associated with aberrant TTK levels (e.g.,associated with overexpression or overproduction of TTK), e.g. byinhibition of gene product production (e.g., decreasing levels oftranscription and/or translation), by decreasing TTK activity (e.g., bydecreasing TTK gene product production (e.g., at the level oftranscription or translation) and/or by reducing one or more of TTK'skinase activities).

[0074] Various aspects of the invention will now be described in moredetail.

[0075] Diagnostic Methods

[0076] In one aspect the invention is based on the discovery that TTKactivity is present at higher levels in cancerous cells (particularly incolon cancer and breast cancer) than in normal cells of the same celltype. This discovery serves as the basis for identification of cancerouscells, as well as identification of tumors that are susceptible totherapy by inhibiting activity of TTK, e.g., by inhibiting TTKexpression at the level of transcription or translation or both, byinhibiting TTK activity, and the like.

[0077] TTK gene products e.g. TTK encoding mRNA or TTK polypeptides areof particular interest as markers (e.g., in bodily fluids (such asblood) or in tissues) to detect the earliest changes along thecarcinogenesis pathway (e.g., to differentiate cancerous tissue fromnon-cancerous tissue) and/or to monitor the efficacy of varioustherapies and preventive interventions. For example, a relativelyincreased level of expression of TTK compared to normal cells or tissuesof the same type can be indicative of a poorer prognosis, and thereforewarrant more aggressive therapy (e.g., chemo- or radio-therapy) for apatient or vice versa. The correlation of surrogate tumor specificfeatures with response to treatment and outcome in patients can defineprognostic indicators that allow the design of tailored therapy based onthe molecular profile of the tumor. These therapies include antibodytargeting, antagonists (e.g., small molecules), and gene therapy.Determining TTK expression and comparison of a patient's profile withknown expression in normal tissue and variants of the disease allows adetermination of the best possible treatment for a patient, both interms of specificity of treatment and in terms of comfort level of thepatient. Surrogate tumor markers, such as polynucleotide expression, canalso be used to better classify, and thus diagnose and treat, differentforms and disease states of cancer. Two classifications widely used inoncology that can benefit from identification of TTK expression levelsare staging of the cancerous disorder, and grading the nature of thecancerous tissue.

[0078] TTK polynucleotides, as well as their encoded gene products, canbe useful to monitor patients having or susceptible to cancer to detectpotentially malignant events at a molecular level before they aredetectable at a gross morphological level. In addition, detection of TTKgene products can be useful as therametrics, e.g., to assess theeffectiveness of therapy by using the polynucleotides or their encodedgene products, to assess, for example, tumor burden in the patientbefore, during, and after therapy.

[0079] Furthermore, a polynucleotide identified as corresponding to agene that is differentially expressed in, and thus is important for, onetype of cancer can also have implications for development or risk ofdevelopment of other types of cancer, e.g., where a polynucleotiderepresents a gene differentially expressed across various cancer types.Thus, for example, expression of a polynucleotide corresponding to agene that has clinical implications for metastatic colon cancer can alsohave clinical implications for stomach cancer or endometrial cancer.

[0080] In making a diagnosis, prognosis, risk assessment, or measurementof tumor burden based on the enzymatic activity of TTK or the expressionlevels of TTK polypeptide or TTK encoding polynucleotides, activity orexpression levels may be compared to those of suitable cancerous ornon-cancerous control samples. For example, a diagnosis of cancer can bemade if TTK activity is increased at by 25%, 50%, 75%, 90%, up to 100%,or, alternatively by 5-fold, 10-fold, 50-fold, or more than 100-foldrelative to a normal non-cancerous cell of the same tissue type.

[0081] Other gene products that are differentially expressed incancerous cells relative to, for example, non-cancer cells of betweencancer cells of differing malignant potential (e.g., non-malignant tumorcells versus cells of high potential malignancy) can also be assayed inaddition to TTK for differential expression in a test cell. Suchexemplary gene products include, but are not necessarily limited toMAPKAP kinase 2 (SEQ ID. No. 33 and 34), MARCKS (SEQ ID NO:35 and 36)and/or IGF2 (SEQ ID NO:37 and 38).

[0082] Staging. Staging is a process used by physicians to describe howadvanced the cancerous state is in a patient. Staging assists thephysician in determining a prognosis, planning treatment and evaluatingthe results of such treatment. Staging systems vary with the types ofcancer, but generally involve the following “TNM” system: the type oftumor, indicated by T; whether the cancer has metastasized to nearbylymph nodes, indicated by N; and whether the cancer has metastasized tomore distant parts of the body, indicated by M. Generally, if a canceris only detectable in the area of the primary lesion without havingspread to any lymph nodes it is called Stage I. If it has spread only tothe closest lymph nodes, it is called Stage II. In Stage II, the cancerhas generally spread to the lymph nodes in near proximity to the site ofthe primary lesion. Cancers that have spread to a distant part of thebody, such as the liver, bone, brain or other site, are Stage IV, themost advanced stage.

[0083] The differential expression level of TTK can facilitatefine-tuning of the staging process by identifying markers for theaggressiveness of a cancer, e.g. the metastatic potential, as well asthe presence in different areas of the body. Thus, a Stage II cancerwith a large differential level of expression of TTK can signify acancer with a high metastatic potential and can be used to change aborderline Stage II tumor to a Stage III tumor, justifying moreaggressive therapy.

[0084] Grading of cancers. Grade is a term used to describe how closelya tumor resembles normal tissue of its same type. The microscopicappearance of a tumor is used to identify tumor grade based onparameters such as cell morphology, cellular organization, and othermarkers of differentiation. As a general rule, the grade of a tumorcorresponds to its rate of growth or aggressiveness, withundifferentiated or high-grade tumors generally being more aggressivethan well differentiated or low-grade tumors. The following guidelinesare generally used for grading tumors: 1) GX Grade cannot be assessed;2) G1 Well differentiated; G2 Moderately well differentiated; 3) G3Poorly differentiated; 4) G4 Undifferentiated. TTK activity levels(e.g., expression levels) can be especially valuable in determining thegrade of the tumor, as they not only can aid in determining thedifferentiation status of the cells of a tumor, they can also identifyfactors other than differentiation that are valuable in determining theaggressiveness of a tumor, such as metastatic potential.

[0085] Detection of colon cancer. Polynucleotides and polypeptidescorresponding to TTK can be used to detect colon cancer in a subject.Colorectal cancer is one of the most common neoplasms in humans andperhaps the most frequent form of hereditary neoplasia. Prevention andearly detection are key factors in controlling and curing colorectalcancer. Colorectal cancer begins as polyps, which are small, benigngrowths of cells that form on the inner lining of the colon. Over aperiod of several years, some of these polyps accumulate additionalmutations and become cancerous. Multiple familial colorectal cancerdisorders have been identified, which are summarized as follows: 1)Familial adenomatous polyposis (FAP); 2) Gardner's syndrome; 3)Hereditary nonpolyposis colon cancer (HNPCC); and 4) Familial colorectalcancer in Ashkenazi Jews. The expression of appropriate polypeptideandpolynucleotides can be used in the diagnosis, prognosis andmanagement of colorectal cancer. Detection of colon cancer can bedetermined using expression levels of TTK alone or in combination withthe levels of expression of other genes differentially expressed incolon cancer. Determination of the aggressive nature and/or themetastatic potential of a colon cancer can be determined by comparinglevels of TTK with a level associated with a normal cell, and comparingtotal levels of another sequence known to be differentially expressed,or otherwise be a marker of, cancerous tissue, e.g., expression of p53,DCC, ras, FAP (see, e.g., Fearon E R, et al., Cell (1990) 61(5):759;Hamilton S R et al., Cancer (1993) 72:957; Bodmer W, et al., Nat Genet.(1994) 4(3):217; Fearon E R, Ann N Y Acad Sci. (1995) 768:101)or MAPKAPkinase 2 (SEQ ID. No. 33 and 34), MARCKS (SEQ ID NO:35 and 36) and/orIGF2 (SEQ ID NO:37 and 38). For example, development of colon cancer canbe detected by examining the level of expression of a gene correspondingto a polynucleotides described herein to the levels of oncogenes (e.g.ras) or tumor suppressor genes (e.g. FAP or p53). Thus expression ofspecific marker polynucleotides can be used to discriminate betweennormal and cancerous colon tissue, to discriminate between colon cancerswith different cells of origin, to discriminate between colon cancerswith different potential metastatic rates, etc. For a review of markersof cancer, see, e.g., Hanahan et al. (2000) Cell 100:57-70.

[0086] Detection of breast cancer. The majority of breast cancers areadenocarcinomas subtypes, which can be summarized as follows: 1) ductalcarcinoma in situ (DCIS), including comedocarcinoma; 2) infiltrating (orinvasive) ductal carcinoma (IDC); 3) lobular carcinoma in situ (LCIS);4) infiltrating (or invasive) lobular carcinoma (ILC); 5) inflammatorybreast cancer; 6) medullary carcinoma; 7) mucinous carcinoma; 8) Paget'sdisease of the nipple; 9) Phyllodes tumor; and 10) tubular carcinoma.

[0087] The expression levels of TTK can be used in the diagnosis andmanagement of breast cancer, as well as to distinguish between types ofbreast cancer. Detection of breast cancer can be determined usingexpression levels of TTK, either alone or in combination with expressionof other gene known to be differentially expressed in breast cancer.Determination of the aggressive nature and/or the metastatic potentialof a breast cancer can also be determined by comparing levels of TTK andcomparing levels of another sequence known to vary in cancerous tissue,e.g. ER expression. In addition, development of breast cancer can bedetected by examining the ratio of expression of TTK to the levels ofsteroid hormones (e.g., testosterone or estrogen) or to other hormones(e.g., growth hormone, insulin). Thus expression of specific markerpolynucleotides and polypeptides can be used to discriminate betweennormal and cancerous breast tissue, to discriminate between breastcancers with different cells of origin, to discriminate between breastcancers with different potential metastatic rates, etc.

[0088] Detection Methods

[0089] A number of methods are known in the art for analyzing biologicalsamples from individuals to determine whether the individual hasincreased expression of a TTK gene product (e.g., RNA or protein) bydetecting the TTK gene product in a biological sample from that subject.As discussed above, the purpose of such analysis may be used fordiagnosis, to detect the presence of an existing cancer, to helpidentify the type of cancer, to assist a physician in determining theseverity or likely course of the cancer, and/or to optimize treatment ofit. In specific non-limiting embodiments, the methods are useful fordetecting cancer cells, facilitating diagnosis of cancer and theseverity of a cancer (e.g., tumor grade, tumor burden, and the like) ina subject, facilitating a determination of the prognosis of a subject,and assessing the responsiveness of the subject to therapy (e.g., byproviding a measure of therapeutic effect through, for example,assessing tumor burden during or following a chemotherapeutic regimen).In additional embodiments, the methods are useful for classification orstratification of cancer cells, e.g., for the purpose of selectingpatients to be included in a clinical trial population, for selecting anappropriate therapy (e.g., selecting therapy according to an expressionprofile of the cancerous cells), and the like.

[0090] Kits

[0091] The detection methods can be provided as part of a kit. Thus, theinvention further provides kits for detecting the presence and/or alevel of TTK activity e.g., by detection of a TTK-encoding mRNA and/or apolypeptide encoded thereby or by measuring TTK activity, in abiological sample. Procedures using these kits can be performed byclinical laboratories, experimental laboratories, medical practitioners,or private individuals. The kits of the invention for detecting TTKpolypeptide that is differentially expressed in cancer cells comprise amoiety that specifically binds the polypeptide, which may be a specificantibody. The kits of the invention for detecting a TTK-encodingpolynucleotide that is differentially expressed in cancer cells comprisea moiety that specifically hybridizes to such a polynucleotide such as aprimer. The kits of the invention for detecting TTK activity comprise arecipient substrate capable of being phosphorylated by TTK, and alabeled donor substrate. The kits may optionally provide additionalcomponents that are useful in the procedure, including, but not limitedto, buffers, developing reagents, labels, reacting surfaces, means fordetection, control samples, standards, instructions, and interpretiveinformation.

[0092] Screening for TTK Nucleic Acid or Polypeptide

[0093] Methods for detection of TTK activity include screening for thepresence of TTK nucleic acid sequences representing an expressed TTKgene or alleles or variants thereof, and detecting the TTK polypeptide.The methods make use of biological samples from individuals that aresuspected of contain the nucleic acid sequences or polypeptide. Examplesof biological samples include blood, plasma, serum, tissue samples,tumor samples, saliva and urine.

[0094] Exemplary approaches for detecting TTK nucleic acid orpolypeptides include: (a) determining the presence of the polypeptideencoded by the TTK gene; (b) using a specific binding member capable ofbinding to a TTK nucleic acid sequence (e.g., a known complementarysequence), the specific binding member comprising a nucleic acid thathybridizes with the TTK sequence under stringent conditions (c) using asubstance comprising an antibody domain with specificity for a TTKnucleic acid sequence or the polypeptide encoded by it, the specificbinding member being labeled to allow detection of the specific bindingmember to its binding partner is detectable; (d) using PCR involving oneor more primers to determine relative levels of TTK in a sample from apatient; and (e) using an assay for TTK activity, e.g., phosphorylationof a TTK substrate.

[0095] The determination of TTK levels can include both levels of normalTTK and/or variant forms of TTK. A variant form of the gene may containone or more insertions, deletions, substitutions and/or additions of oneor more nucleotides compared with the wild-type sequence which may ormay not alter the gene function. Differences at the nucleic acid levelare not necessarily reflected by a difference in the amino acid sequenceof the encoded polypeptide due to the degeneracy of the genetic code.However, a mutation or other difference in a gene may result in aframe-shift or stop codon, which could seriously affect the nature ofthe polypeptide produced (if any), or a point mutation or grossmutational change to the encoded polypeptide, including insertion,deletion, substitution and/or addition of one or more amino acids orregions in the polypeptide.

[0096] A mutation in a promoter sequence or other regulatory region mayalter (e.g., reduce or enhance) expression from the gene or affect theprocessing or stability of the mRNA transcript.

[0097] There are various methods for detecting a particular nucleic acidsequence in a test sample. Tests may be carried out on preparationscontaining mRNA or cDNA generated from isolated mRNA in a manner thatreflects the relative levels of mRNA transcripts in the sample. Levelsof RNA can be determined specific amplification reaction such as PCRusing one or more pairs of primers may be employed to amplify a regionof the nucleic acid, and preferably a region with less homology to othergenes. Nucleic acid for testing may be prepared from nucleic acidremoved from cells or in a library using a variety of other techniquessuch as restriction enzyme digest and electrophoresis.

[0098] Nucleic acid may be screened using a TTK-specific probe. Such aprobe corresponds in sequence to a region of the TTK gene, or itscomplement. Under stringent conditions, specific hybridization of such aprobe to test nucleic acid is indicative of the presence of the TTKnucleic acid in a sample. For efficient screening purposes, more thanone probe may be used on the same test sample. The probe may contain asfew as 15, 20, 50 or 100 nucleotides of the TTK gene of SEQ ID. No. 13or may be as long as or 500, 1 kb or as much as 3.8 kb or longer inlength.

[0099] Allele- or variant-specific oligonucleotides may similarly beused in PCR to specifically amplify particular sequences if present in atest sample. Assessment of whether a PCR band contains a gene variantmay be carried out in a number of ways familiar to those skilled in theart. The PCR product may for instance be treated in a way that enablesone to display the mutation or polymorphism on a denaturingpolyacrylamide DNA sequencing gel, with specific bands that are linkedto the gene variants being selected. This can be done simultaneous to orsequentially to determining the level of a normal TTK sequence, e.g., todetermine the combinatory levels of total TTK.

[0100] The presence of absence of a lesion in a promoter or otherregulatory sequence may also be assessed by determining the level ofmRNA production by transcription or the level of polypeptide productionby translation from the mRNA. The presence of differences in sequence ofnucleic acid molecules may be detected by means of restriction enzymedigestion, such as in a method of DNA fingerprinting where therestriction pattern produced when one or more restriction enzymes areused to cut a sample of nucleic acid is compared with the patternobtained when a sample containing the normal gene or a variant or alleleis digested with the same enzyme or enzymes.

[0101] A test sample of nucleic acid may be provided for example byextracting nucleic acid from cells, e.g., cells from a tumor biopsy.

[0102] Detection of TTK Polypeptides

[0103] There are various methods for determining the presence or absencein a test sample of a TTK polypeptide. A sample may be tested for thepresence of a binding partner for a specific binding member such as anantibody (or mixture of antibodies), specific for wild-type TTK and/orone or more particular variants (e.g., allelic variants) of the TTKpolypeptide. In such cases, the sample may be tested by being contactedwith a specific binding member such as an antibody under appropriateconditions for specific binding. Where a panel of antibodies is used,different reporting labels may be employed for each antibody so thatbinding of each can be determined. In addition to detection of TTKpolypeptides using anti-TTK antibodies, TTK polypeptide can also beidentified using TTK-specific activity assays.

[0104] Arrays

[0105] Binding agents (such as antibodies or nucleic acid sequences) canalso be immobilized in small, discrete locations and/or as arrays onsolid supports or on diagnostic chips. These approaches can beparticularly valuable as they can provide great sensitivity,particularly through the use of fluorescently labeled reagents, requireonly very small amounts of biological sample from individuals beingtested and allow a variety of separate assays can be carried outsimultaneously. This latter advantage can be useful as it provides anassay for different proteins (e.g., an oncogene or tumor suppressor) intandem with the assay for TTK. Thus, in a further aspect, the presentinvention provides a support or diagnostic chip having immobilizedthereon one or more binding agents capable of specifically binding TTKnucleic acid or polypeptides, optionally in combination with otherreagents needed to carrying out an assay.

[0106] Methods for Expression of TTK Polypeptide

[0107] The full-length or partial polypeptides encoded by TTK may beexpressed in any expression system, including, for example, bacterial,yeast, insect, amphibian and mammalian systems. Suitable vectors andhost cells for which are described in U.S. Pat. No. 5,654,173.Appropriate polynucleotide constructs are purified using standardrecombinant DNA techniques as described in, for example, Sambrook etal., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold SpringHarbor Press, Cold Spring Harbor, N.Y.), and under current regulationsdescribed in United States Dept. of HHS, National Institute of Health(NIH) Guidelines for Recombinant DNA Research.

[0108] Bacteria. Expression systems in bacteria include those describedin Chang et al., Nature (1978) 275:615, Goeddel et al., Nature (1979)281:544, Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776,U.S. Pat. No. 4,551,433, DeBoer et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:21-25, and Siebenlist et al., Cell (1980) 20:269.

[0109] Yeast. Expression systems in yeast include those described inHinnen et al., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al.,J. Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986)6:142; Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al.,J. Gen. Microbiol. (1986) 132:3459, Roggenkamp et al., Mol. Gen. Genet.(1986) 202:302) Das et al., J. Bacteriol. (1984) 158:1165; DeLouvencourt et al., J. Bacteriol. (1983) 154:737, Van den Berg et al.,Bio/Technology (1990) 8:135; Kunze et al., J. Basic Microbiol. (1985)25:141; Cregg et al., Mol. Cell. Biol. (1985) 5:3376, U.S. Pat. Nos.4,837,148 and 4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidowet al., Curr. Genet. (1985) 10:380, Gaillardin et al., Curr. Genet.(1985) 10:49, Ballance et al., Biochem. Biophys. Res. Commun. (1983)112:284-289; Tilburn et al., Gene (1983) 26:205-221, Yelton et al.,Proc. Natl. Acad. Sci. (USA) (1984) 81:1470-1474, Kelly and Hynes, EMBOJ. (1985) 4:475479; EP 0 244,234, and WO 91/00357.

[0110] Insect Cells. Expression of heterologous genes in insects isaccomplished as described in U.S. Pat. No. 4,745,051, Friesen et al.(1986) “The Regulation of Baculovirus Gene Expression” in: The MolecularBiology Of Baculoviruses (W. Doerfler, ed.), EP 0 127,839, EP 0 155,476,and Vlak et al., J. Gen. Virol. (1988) 69:765-776, Miller et al., Ann.Rev. Microbiol. (1988) 42:177, Carbonell et al., Gene (1988) 73:409,Maeda et al., Nature (1985) 315:592-594, Lebacq-Verheyden et al., Mol.Cell Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad. Sci. (USA)(1985) 82:8404, Miyajima et al., Gene (1987) 58:273; and Martin et al.,DNA (1988) 7:99. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts are described inLuckow et al., Bio/Technology (1988) 6:47-55, Miller et al., GenericEngineering (Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing,1986), pp. 277-279, and Maeda et al., Nature, (1985) 315:592-594.

[0111] Mammalian Cells. Mammalian expression is accomplished asdescribed in Dijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc.Natl. Acad. Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985)41:521 and U.S. Pat. No. 4,399,216. Other features of mammalianexpression are facilitated as described in Ham and Wallace, Meth. Enz.(1979) 58:44, Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat.Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO87/00195, and U.S. RE 30,985.

[0112] Screening Assays to Identify Chemotherapeutic Agents

[0113] The invention also encompasses screening assays to identifyagents that modulate TTK activity, specifically that decrease aberrantTTK activity in an affected cell, e.g., a cancerous or pre-cancerouscell in which TTK is differentially expressed. Such assays may beperformed either in vitro or in vivo.

[0114] Candidate Agents

[0115] The term “agent” as used herein describes any molecule with thecapability of altering the expression or physiological function of agene product of a differentially expressed gene. Generally a pluralityof assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration or below the level ofdetection.

[0116] Candidate agents encompass numerous chemical classes, including,but not limited to, organic molecules (e.g., small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons), peptides, monoclonal antibodies, antisense polynucleotides,and ribozymes, and the like. Candidate agents can comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including, but not limited to:polynucleotides, peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

[0117] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Candidate agents can be assessed for modulation ofTTK activity either singly or in pools.

[0118] Screening of Candidate Agents In Vitro

[0119] A wide variety of in vitro assays may be used to screen candidateagents for the desired biological activity, including, but not limitedto, labeled in vitro protein-protein binding assays, protein-DNA bindingassays (e.g., to identify agents that affect expression),electrophoretic mobility shift assays, immunoassays for protein binding,and the like. For example, by providing for the production of largeamounts of a differentially expressed polypeptide, one can identifyligands or substrates that bind to, modulate or mimic the action of thepolypeptide. Further methods for identifying these ligands andsubstrates are provided below. The purified polypeptide may also be usedfor determination of three-dimensional crystal structure, which can beused for modeling intermolecular interactions, transcriptionalregulation, etc.

[0120] The screening assay can be a binding assay, wherein one or moreof the molecules may be joined to a label, and the label directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g.,magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

[0121] A variety of other reagents may be included in the screeningassays described herein. Where the assay is a binding assay, theseinclude reagents like salts, neutral proteins, e.g.,albumin, detergents,etc that are used to facilitate optimal protein-protein binding,protein-DNA binding, and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used. The mixture of components are added in any order thatprovides for the requisite binding. Incubations are performed at anysuitable temperature, typically between 4 and 40° C. Incubation periodsare selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 and 1hours will be sufficient.

[0122] Many mammalian genes have homologs in yeast and lower animals.The study of such homologs physiological role and interactions withother proteins in vivo or in vitro can facilitate understanding ofbiological function. In addition to model systems based on geneticcomplementation, yeast has been shown to be a powerful tool for studyingprotein-protein interactions through the two hybrid system described inChien et al. 1991 Proc. Natl. Acad. Sci. USA 88:9578-9582.

[0123] Screening of Candidate Agents In Vivo

[0124] Candidate agents can be screened in a non-human animal model ofcancer (e.g., in animals into which have been injected cancerous cells;in animals that are transgenic for an alteration in expression of adifferentially expressed gene as described herein, e.g., a transgenic“knock-out,” or a transgenic “knock-in,” a polynucleotide encoding allor a portion of a differentially expressed gene product and comprisingan operably linked reporter gene, and the like).

[0125] In general, the candidate agent is administered to the animal,and the effects of the candidate agent determined. The candidate agentcan be administered in any manner desired and/or appropriate fordelivery of the agent in order to effect a desired result. For example,the candidate agent can be administered by injection (e.g., by injectionintravenously, intramuscularly, subcutaneously, or directly into thetissue in which the desired affect is to be achieved), orally, or by anyother desirable means. Normally, the in vivo screen will involve anumber of animals receiving varying amounts and concentrations of thecandidate agent (from no agent to an amount of agent hat approaches anupper limit of the amount that can be delivered successfully to theanimal), and may include delivery of the agent in different formulation.The agents can be administered singly or can be combined in combinationsof two or more, especially where administration of a combination ofagents may result in a synergistic effect.

[0126] The effect of agent administration upon the transgenic animal canbe monitored by assessing expression of the gene product, growth of theinjected tumor cells, and the like.

[0127] Identified Candidate Agents

[0128] Compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of a condition that is amenable to treatment by modulation ofexpression of a differentially expressed gene product. The therapeuticagents may be administered in a variety of ways, orally, topically,parenterally e.g., subcutaneously, intraperitoneally, by viralinfection, intravascularly, etc. Oral and inhaled treatments are ofparticular interest. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways. The concentration oftherapeutically active compound in the formulation may vary from about0.1-100 wt. %. The therapeutic agents can be administered in a singledose, or as multiple doses over a course of treatment.

[0129] The pharmaceutical compositions can be prepared in various forms,such as granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

[0130] Methods of Screening for Drugs that Modulate TTK Activity

[0131] A TTK polypeptide or TTK-encoding nucleic acid according to thepresent invention may be used in screening for molecules which affect ormodulate TTK activity or function. Such molecules may be useful in atherapeutic and/or prophylactic context. Means for screening forsubstances potentially useful in treating or preventing cancer isprovided by the present invention. In general, the methods of theinvention are to facilitate identification of modulators of TTK activity(e.g., by modulating activity of TTK polypeptide or other TTK geneproduct, or by affecting TTK activity by targeting activity of geneproducts that act either upstream or downstream of TTK in a cascade thatleads to TTK activity), with agents that decrease TTK activity generallybeing of particular interest. Substances identified as modulators of theTTK activity represent an advance in the fight against cancer since theyprovide basis for design and investigation of pharmaceuticals for invivo use.

[0132] A method of screening for a substance which modulates activity ofa polypeptide may include contacting one or more test substances withthe polypeptide in a suitable reaction medium, testing the activity ofthe treated polypeptide (e.g., the ability to phosphorylate itssubstrate) and comparing that activity with the activity of thepolypeptide in comparable reaction medium untreated with the testsubstance or substances. A difference in activity between the treatedand untreated polypeptides is indicative of a modulating effect of therelevant test substance or substances.

[0133] Combinatorial library technology provides an efficient way oftesting a potentially vast number of different substances for ability tomodulate activity of a polypeptide. Such libraries and their use areknown in the art. The use of peptide libraries is preferred. Testsubstances may also be screened for ability to interact with thepolypeptide, e.g., in a yeast two-hybrid system. This may be used as acoarse screen prior to testing a substance for actual ability tomodulate activity of the polypeptide. Alternatively, the screen could beused to screen test substances for binding to a TTK specific bindingpartner.

[0134] A substance identified using as a modulator of TTK polypeptidefunction may be peptide or non-peptide in nature. Non-peptide “smallmolecules” are often preferred for many in vivo pharmaceutical uses.Accordingly, a mimetic or mimic of the substance (particularly if apeptide) may be designed for pharmaceutical use.

[0135] TTK activity assays

[0136] The activity of the TTK may be measured using any suitable kinaseassay known in the art. For example, and not by way of limitation, themethods described in Hogg et al (Oncogene 1994 9:98-96), Mills et al (J.Biol. Chem. 1992 267:16000-006) and Tomizawa et al 2001 (FEBS Lett. 2001492: 221-7), Schmandt et al, (J. Immunol. 1994, 152:96-105) may be used.Further serine, threonine and tyrosine kinase assays are described inAusubel et al. (Short Protocols in Molecular Biology, 1999, unit 17.6).

[0137] TTK assays generally use TTK polypeptide, a labeled donorsubstrate, and a receptor substrate that is either specific ornon-specific for TTK. In such assays TTK transfers a labeled moiety fromthe donor substrate to the receptor substrate, and kinase activity ismeasured by the amount of labeled moiety transferred from the donorsubstrate to the receptor substrate.

[0138] TTK polypeptide may be produced using various expression systemsas detailed above, may be purified from cells, may be in the form of acleaved or uncleaved recombinant fusion protein and may have non-TTKpolypeptide sequences, for example a His tag or β-galactosidase at itsN- or C-terminus. TTK activity may be assayed in cancerous cells linesif the cancerous cell lines are used as a source of the TTK to beassayed. Suitable donor substrates for TTK assays include any moleculethat is susceptible to dephosphorylation by TTK include γ-labeled ATPand ATP analogs, wherein the label is ³³P, ³²P, ³⁵S or any otherradioactive isotope or a suitable fluorescent marker. Suitable recipientsubstrates for TTK assays include any polypeptide or other molecule thatis susceptible to phosphorylation by TTK. Recipient substrates areusually derived from fragments of in vivo targets of TTK. Recipientsubstrates fragments may be 8 to 50 amino acids in length, usually 10 to30 amino acids and preferably of about 10, 12, 15, 18, 20 and 25 aminoacids in length Further recipient substrates can be determinedempirically using a set of different polypeptides or other molecules.Targets of TTK suitable for TTK assays include tau and cdc25. Recipientsubstrates for TTK are typically capable of being purified from othercomponents of the reaction once the reaction has been performed. Thispurification is usually done through a molecular interaction, where therecipient substrates is biotinylated and purified through itsinteraction with streptavidin, or a specific antibody is available thatcan specifically recognize the recipient substrates. The reaction can beperformed in a variety of conditions, such as on a solid support, in agel, in solution or in living cells.

[0139] One exemplary recipient substrate for TTK phosphorylation is thehuman protein cdc25, SEQ ID NO:26, which is phosphorylated by TTK at theserine residues of amino acid position 214 and 216. Two fragments ofcdc25 are used as substrates in the kinase assay described below. Thesefragments comprise peptides A (SEQ ID NO:27), corresponding to aminoacids 209 to 225 of the cdc25 polypeptide sequence or peptide B (SEQ IDNO:28), corresponds to amino acids 210 to 223 of the cdc25 polypeptide.In this assay, two biotinylated polypeptides of comprising either SEQ IDNO:27 (Biotin-SGSGSGLYRSPSMPENLNRPR-NH2) or SEQ ID NO:28(Biotin-GGGGLYRSPSMPENLNRK-OH) are used.

[0140] The choice of detection methods depends on type of label used forthe donor molecule and may include, for example, measurement ofincorporated radiation or fluorescence by autoradiography,scintillation, scanning or fluorography.

[0141] Methods of Inhibiting Tumor Growth and Other Treatment Goals

[0142] The invention further provides methods for reducing growth ofcancer cells, particular breast or colon cancer cells. In general, themethods comprise contacting a cancer cell that expresses TTK at anaberrant level relative to normal cells with a substance that (1)modulates, generally decreases, expression of TTK (e.g., a antisensepolynucleotide corresponding to TTK); or (2) otherwise modulates,generally decreases, TTK polypeptide levels and/or TTK activity in acancerous cell having aberrant TTK activity.

[0143] “Reducing growth of a cancer cell” includes, but is not limitedto, reducing proliferation of cancer cells, and reducing the incidenceof a normal cell from developing a cancerous phenotype or morphology.Whether a reduction in cancer cell growth has been achieved can bereadily determined using any known assay, including, but not limited to,[³H]-thymidine incorporation; counting cell number over a period oftime; detecting, measuring a marker associated with colon cancer (e.g.,CEA, CA19-9, and LASA), and/or methods well known in the art forassessing tumor burden.

[0144] The present invention provides methods for treating cancer(particularly breast and colon cancer or other cancer that is associatedwith aberrantly high TTK activity) which methods generally compriseadministering to an individual an agent that reduces TTK activity in anamount sufficient to reduce cancer cell growth to treat the cancer.Whether a substance, or a specific amount of the substance, is effectivein treating cancer can be assessed using any of a variety of knowndiagnostic assays, e.g. in the case of colon cancer, sigmoidoscopy,proctoscopy, rectal examination, colonoscopy with biopsy, contrastradiographic studies, CAT scans, angiography, and detection of a tumormarker associated with colon cancer in the blood of the individual. Thesubstance can be administered systemically or locally. Thus, in someembodiments, the substance is administered locally, and colon cancergrowth is decreased at the site of administration. Local administrationmay be useful in treating, e.g., a solid tumor.

[0145] In one embodiment, the invention features polynucleotides thatact as antisense polynucleotides and decrease TTK activity. AntisenseTTK polynucleotides generally comprise a polynucleotide of at leastabout 20 to 3000 nucleotides, usually at least about 20 to 1000nucleotides and more usually at least about 8 to 50 nucleotides, andpreferably about 26, 20, 18, 17, 15, 10 and 8 nucleotides. Exemplary TTKpolynucleotides are provided in the Examples and in SEQ ID NO:1-12,although any antisense fragment of SEQ ID NO:13 will suffice.

[0146] The therapeutic regimen is selected according to the expressionprofile. For example, if a patient's tumor indicates that the tumorproduces aberrantly high level of TTK relative to normal cells, then adrug having efficacy in the treatment of such TTK-expressing tumors isselected for therapy of that patient.

[0147] Pharmaceutical Compositions

[0148] Pharmaceutical compositions of the invention can comprise atherapeutically effective amount of a polypeptide, antibody,polynucleotide (including antisense nucleotides and ribozymes), or smallmolecule or other compound identified as modulating activity of TTK,preferably decreasing TTK activity. The term “therapeutically effectiveamount” as used herein refers to an amount of a therapeutic agent totreat, ameliorate, or prevent a desired disease or condition, or toexhibit a detectable therapeutic or preventative effect. The effect canbe detected by, for example, chemical markers or antigen levels.Therapeutic effects also include reduction in physical symptoms, such asdecreased body temperature, and/or in the effect upon tumor load in thesubject (e.g., decrease in tumor size or inhibition in tumor growth).The precise effective amount for a subject will depend upon thesubject's size and health, the nature and extent of the condition, andthe therapeutics or combination of therapeutics selected foradministration. Thus, it is not useful to specify an exact effectiveamount in advance. However, the effective amount for a given situationis determined by routine experimentation and is within the judgment ofthe clinician. For purposes of the present invention, an effective dosewill generally be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg toabout 10 mg/kg of the DNA constructs in the individual to which it isadministered.

[0149] A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, and other therapeutic agents. Theterm refers to any pharmaceutical carrier that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, and which can be administered without undue toxicity.Suitable carriers can be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Pharmaceutically acceptable carriers in therapeuticcompositions can include liquids such as water, saline, glycerol andethanol. Auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, can also be present in suchvehicles. Typically, the therapeutic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection can also be prepared. Liposomes are included within thedefinition of a pharmaceutically acceptable carrier. Pharmaceuticallyacceptable salts can also be present in the pharmaceutical composition,e.g., mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. A thoroughdiscussion of pharmaceutically acceptable excipients is available inRemington 's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Theprecise nature of the carrier or other material may depend on the routeof administration, e.g., oral, intravenous, cutaneous or subcutaneous,nasal, intramuscular, intraperitoneal routes.

[0150] Pharmaceutical compositions for oral administration may be intablet, capsule, powder or liquid form. A tablet may include a solidcarrier such as gelatin or an adjuvant. Liquid pharmaceuticalcompositions generally include a liquid carrier such as water,petroleum, animal or vegetable oils, mineral oil or synthetic oil.Physiological saline solution, dextrose or other saccharide solution orglycols such as ethylene glycol, propylene glycol or polyethylene glycolmay be included.

[0151] For intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which is hassuitable pH, isotonicity and stability. Suitable solutions, for example,optionally include but are not limited to isotonic vehicles such assodium chloride, preservatives, stabilizers, buffers, antioxidantsand/or other additives as required.

[0152] Administration of the pharmaceutical is administered in aprophylactically effective amount or a therapeutically effective amount.The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated. Decisions on dosage etc, can be determined by one skilled inthe art based upon the disclosed methods, and typically takes account ofthe disorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners. Examples of the techniques and protocols mentionedabove can be found in Remington's Pharmaceutical Sciences, 16th edition,Osol, A. (ed), 1980.

[0153] Alternatively, targeting therapies may be used to deliver theactive agent more specifically to certain types of cell, by the use oftargeting systems such as antibody or cell specific ligands. Targetingmay be desirable for a variety of reasons; for example if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.Targeting can be accomplished by, for example, administering adrug-antibody complex to a subject, wherein the antibody is specific fora cancer-associated antigen, and the drug is one that reduces cancercell growth. Targeting can be accomplished by coupling (e.g., linking,directly or via a linker molecule, either covalently or non-covalently,so as to form a drug-antibody complex) a drug to an antibody specificfor a cancer-associated antigen. Methods of coupling a drug to anantibody are well known in the art and need not be elaborated uponherein.

[0154] Pharmaceutical agents can also be produced in the target cells byexpression from an encoding gene introduced into the cells, e.g., in aviral or liposomal vector. The vector could be targeted to the specificcells to be treated, or it could contain regulatory elements which areswitched on more or less selectively by the target cells.

[0155] Alternatively, the agent could be administered in a precursorform, for conversion to the active form by an activating agent producedin, or targeted to, the cells to be treated. A composition may beadministered alone or in combination with other treatments, eithersimultaneously or sequentially dependent upon the condition to betreated.

[0156] Delivery Methods for Therapy

[0157] Once formulated, the compositions of the invention or identifiedusing the methods of the invention can be administered directly to thesubject (e.g., as polynucleotide or polypeptides). Direct delivery ofthe compositions will generally be accomplished by parenteral injection,e.g., subcutaneously, intraperitoneally, intravenously orintramuscularly, intratumoral or to the interstitial space of a tissue.Other modes of administration include oral and pulmonary administration,suppositories, and transdermal applications, needles, and gene guns orhyposprays. Dosage treatment can be a single dose schedule or a multipledose schedule.

[0158] Once a gene corresponding to a polynucleotide of the inventionhas been found to correlate with a proliferative disorder, such asneoplasia, dysplasia, and hyperplasia, the disorder can be amenable totreatment by administration of a therapeutic agent based on the providedpolynucleotide, corresponding polypeptide or other correspondingmolecule (e.g., antisense, ribozyme, etc.).

[0159] The dose and the means of administration are determined based onthe specific qualities of the therapeutic composition, the condition,age, and weight of the patient, the progression of the disease, andother relevant factors. For example, administration of polynucleotidetherapeutic compositions agents of the invention includes local orsystemic administration, including injection, oral administration,particle gun or catheterized administration, and topical administration.Preferably, the therapeutic polynucleotide composition contains anexpression construct comprising a promoter operably linked to apolynucleotide of at least 12, 15, 17, 18, 22, 25, 30, or 35contiguous-nucleotides of the polynucleotide disclosed herein. Variousmethods can be used to administer the therapeutic composition directlyto a specific site in the body. For example, a small metastatic lesionis located and the therapeutic composition injected several times inseveral different locations within the body of tumor. Alternatively,arteries which serve a tumor are identified, and the therapeuticcomposition injected into such an artery, in order to deliver thecomposition directly into the tumor. A tumor that has a necrotic centeris aspirated and the composition injected directly into the now emptycenter of the tumor. The antisense composition is directly administeredto the surface of the tumor, for example, by topical application of thecomposition. X-ray imaging is used to assist in certain of the abovedelivery methods.

[0160] Receptor-mediated targeted delivery of therapeutic compositionscontaining an antisense polynucleotide, subgenomic polynucleotides, orantibodies to specific tissues can also be used. Receptor-mediated DNAdelivery techniques are described in, for example, Findeis et al.,Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics:Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.)(1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. BiolChem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990)87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol.Factors such as method of action (e.g., for enhancing or inhibitinglevels of the encoded gene product) and efficacy of transformation andexpression are considerations which will affect the dosage required forultimate efficacy of the antisense subgenomic polynucleotides. Wheregreater expression is desired over a larger area of tissue, largeramounts of antisense subgenomic polynucleotides or the same amountsreadministered in a successive protocol of administrations, or severaladministrations to different adjacent or close tissue portions of, forexample, a tumor site, may be required to effect a positive therapeuticoutcome. In all cases, routine experimentation in clinical trials willdetermine specific ranges for optimal therapeutic effect. Forpolynucleotide related genes encoding polypeptides or proteins withanti-inflammatory activity, suitable use, doses, and administration aredescribed in U.S. Pat. No. 5,654,173.

[0161] The therapeutic polynucleotides and polypeptides of the presentinvention can be delivered using gene delivery vehicles. The genedelivery vehicle can be of viral or non-viral origin (see generally,Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy(1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt,Nature Genetics (1994) 6:148). Expression of such coding sequences canbe induced using endogenous mammalian or heterologous promoters.Expression of the coding sequence can be either constitutive orregulated.

[0162] Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; U.S. Pat. No. 5, 219,740; WO 93/11230; WO 93/10218; U.S. Pat.No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805),alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forestvirus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCCVR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCCVR-1250; ATCC VR 1249; ATCC VR-532), and adeno-associated virus (AAV)vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938;WO 95/11984 and WO 95/00655). Administration of DNA linked to killedadenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can alsobe employed.

[0163] Non-viral delivery vehicles and methods can also be employed,including, but not limited to, polycationic condensed DNA linked orunlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther.(1992) 3:147); ligand-linked DNA(see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO97/42338) and nucleic charge neutralization or fusion with cellmembranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in WO 90/11092 and U.S. Pat. No.5,580,859. Liposomes that can act as gene delivery vehicles aredescribed in U.S. Pat. No. 5,422,120; WO 95/13796; WO 94/23697; WO91/14445; and EP 0524968. Additional approaches are described in Philip,Mol. Cell Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci.(1994) 91:1581.

[0164] Further non-viral delivery suitable for use includes mechanicaldelivery systems such as the approach described in Woffendin et al.,Proc. Natl. Acad. Sci. USA (1994) 91(24): 11581. Moreover, the codingsequence and the product of expression of such can be delivered throughdeposition of photopolymerized hydrogel materials or use of ionizingradiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033). Otherconventional methods for gene delivery that can be used for delivery ofthe coding sequence include, for example, use of hand-held gene transferparticle gun (see, e.g., U.S. Pat. No. 5,149,655); use of ionizingradiation for activating transferred gene (see, e.g., U.S. Pat. No.5,206,152 and WO 92/11033).

[0165] As an alternative to the use of viral vectors other known methodsof introducing nucleic acid into cells includes electroporation, calciumphosphate co-precipitation, mechanical techniques such asmicroinjection, transfer mediated by liposomes and direct DNA uptake andreceptor-mediated DNA transfer. Gene transfer techniques whichselectively target the TTK nucleic acid to the affected cell type arepreferred. Examples of this included receptor-mediated gene transfer, inwhich the nucleic acid is linked to a protein ligand via polylysine,with the ligand being specific for a receptor present on the surface ofthe target cells.

[0166] Screening for Substances Affecting TTK Expression

[0167] The present invention also provides the use of all or part of thenucleic acid sequence of the TTK promoter and/or enhancer regions inmethods of screening for substances which modulate the activity of thepromoter and increase or decrease the level of TTK expression. Thisassay can be performed to identify anti-cancer agents for therapeuticand/or prophylactic purposes. The level of promoter activity, i.e., theability to initiate transcription, is quantifiable for instance byassessment of the amount of mRNA produced by transcription from thepromoter or by assessment of the amount of protein product produced bytranslation of mRNA produced by transcription from the promoter. Theamount of a specific mRNA present in an expression system may bedetermined for example using specific oligonucleotides which are able tohybridize with the mRNA and which are labeled or may be used in aspecific amplification reaction such as PCR. Use of a reporter genefacilitates determination of promoter activity by reference to proteinproduction.

[0168] Generally, a reporter gene under control of the TTK promoterand/or enhancers may be transcribed into mRNA which may be translatedinto a peptide or polypeptide product which may be detected andpreferably quantitated following expression. The reporter genepreferably encodes an enzyme which catalyses a reaction which produces adetectable signal, preferably a visually detectable signal, such as acoloured product. Many examples are known, including β-galactosidase andluciferase. β-galactosidase activity may be assayed by production ofblue color on substrate, the assay being by eye or by use of aspectrophotometer to measure absorbance. Fluorescence, for example thatproduced as a result of luciferase activity, may be quantitated using aspectrophotometer. Radioactive assays may be used, for instance usingcholoramphenicol acetyltransferase, which may also be used innon-radioactive assays. The presence and/or amount of gene productresulting from expression from the reporter gene may be determined usinga molecule able to bind the product, such as an antibody or fragmentthereof. The binding molecule may be labeled directly or indirectlyusing any standard technique.

[0169] Those skilled in the art are well aware of a multitude ofpossible reporter genes and assay techniques which may be used todetermine gene activity according to the presently disclosed methods.Any suitable reporter/assay may be used and the present invention isintended to encompass such systems.

[0170] Following identification of a substance which modulates oraffects promoter activity, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.manufacture or formulation, of a composition such as a medicament,pharmaceutical composition or drug.

[0171] Integrated Disease Information System

[0172] The levels of TTK in a sample can be used in an integrateddisease information system to aid in analysis such as proposed patientinterventions, designing clinical trials, performing pharmacoeconomicanalysis, and illustrating disease progression for various patients overtime. For example, TTK information determined according to the methodsof the invention can be used in a system such as that described in U.S.Pat. No. 6,108,635 issued to Herren, et al. on Aug. 22, 2000. Such asystem can be for collecting the results of medical treatments given topatients in a plurality of locations. See, e.g., U.S. Pat. No. 5,713,350issued to Yokota, et al. on Feb. 3, 1998.

EXAMPLES

[0173] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Source of Patient Tissue Samples

[0174] Normal and cancerous tissues were collected from patients usinglaser capture microdissection (LCM) techniques, which techniques arewell known in the art (see, e.g., Ohyama et al. (2000) Biotechniques29:530-6; Curran et al. (2000) Mol. Pathol. 53:64-8; Suarez-Quian et al.(1999) Biotechniques 26:328-35; Simone et al (1998) Trends Genet14:272-6; Conia et al. (1997) J. Clin. Lab. Anal. 11:28-38; Emmert-Bucket al. (1996) Science 274:998-1001). Table 1 (inserted following thelast page of the Examples ) provides information about each patient fromwhich the samples were isolated, including: the Patient ID and PathReportID, numbers assigned to the patient and the pathology reports foridentification purposes; the anatomical location of the tumor(AnatomicalLoc); The Primary Tumor Size; the Primary Tumor Grade; theHistopathologic Grade; a description of local sites to which the tumorhad invaded (Local Invasion); the presence of lymph node metastases(Lymph Node Metastasis); incidence of lymph node metastases (provided asnumber of lymph nodes positive for metastasis over the number of lymphnodes examined) (Incidence Lymphnode Metastasis); the Regional LymphnodeGrade; the identification or detection of metastases to sites distant tothe tumor and their location (Distant Met & Loc);a description of thedistant metastases (Description Distant Met); the grade of distantmetastasis (Distant Met Grade); and general comments about the patientor the tumor (Comments). Adenoma was not described in any of thepatients; adenoma dysplasia (described as hyperplasia by thepathologist) was described in Patient ID No. 695. Extranodal extensionswere described in two patients, Patient ID Nos. 784 and 791.Lymphovascular invasion was described in seven patients, Patient ID Nos.128, 278, 517, 534, 784, 786, and 791. Crohn's-like infiltrates weredescribed in seven patients, Patient ID Nos. 52, 264, 268, 392, 393,784, and 791.

Example 2 Differential Expression of TTK

[0175] cDNA probes were prepared from total RNA isolated from thepatient cells described in Example 1. Since LCM provides for theisolation of specific cell types to provide a substantially homogenouscell sample, this provided for a similarly pure RNA sample.

[0176] Total RNA was first reverse transcribed into cDNA using a primercontaining a T7 RNA polymerase promoter, followed by second strand DNAsynthesis. cDNA was then transcribed in vitro to produce antisense RNAusing the T7 promoter-mediated expression (see, e.g., Luo et al. (1999)Nature Med 5:117-122), and the antisense RNA was then converted intocDNA. The second set of cDNAs were again transcribed in vitro, using theT7 promoter, to provide antisense RNA. Optionally, the RNA was againconverted into cDNA, allowing for up to a third round of T7-mediatedamplification to produce more antisense RNA. Thus the procedure providedfor two or three rounds of in vitro transcription to produce the finalRNA used for fluorescent labeling. Fluorescent probes were generated byfirst adding control RNA to the antisense RNA mix, and producingfluorescently labeled cDNA from the RNA starting material. Fluorescentlylabeled cDNAs prepared from the tumor RNA sample were compared tofluorescently labeled cDNAs prepared from normal cell RNA sample. Forexample, the cDNA probes from the normal cells were labeled with Cy3fluorescent dye (green) and the cDNA probes prepared from the tumorcells were labeled with Cy5 fluorescent dye (red).

[0177] Each array used had an identical spatial layout and control spotset. Each microarray was divided into two areas, each area having anarray with, on each half, twelve groupings of 32×12 spots for a total ofabout 9,216 spots on each array. The two areas are spotted identicallywhich provide for at least two duplicates of each clone per array.Spotting was accomplished using PCR amplified products from 0.5 kb to2.0 kb and spotted using a Molecular Dynamics Gen III spotter accordingto the manufacturer's recommendations. The first row of each of the 24regions on the array had about 32 control spots, including 4 negativecontrol spots and 8 test polynucleotides. The test polynucleotides werespiked into each sample before the labeling reaction with a range ofconcentrations from 2-600 pg/slide and ratios of 1:1 . For each arraydesign, two slides were hybridized with the test samples reverse-labeledin the labeling reaction. This provided for about 4 duplicatemeasurements for each clone, two of one color and two of the other, foreach sample.

[0178] The differential expression assay was performed by mixing equalamounts of probes from tumor cells and normal cells of the same patient.The arrays were prehybridized by incubation for about 2 hrs at 60° C. in5×SSC/0.2% SDS/1 mM EDTA, and then washed three times in water and twicein isopropanol. Following prehybridization of the array, the probemixture was then hybridized to the array under conditions of highstringency (overnight at 42° C. in 50% formamide, 5×SSC, and 0.2% SDS.After hybridization, the array was washed at 55° C. three times asfollows: 1) first wash in 1×SSC/0.2% SDS; 2) second wash in 0.1×SSC/0.2%SDS; and 3) third wash in 0.1×SSC.

[0179] The arrays were then scanned for green and red fluorescence usinga Molecular Dynamics Generation III dual color laser-scanner/detector.The images were processed using BioDiscovery Autogene software, and thedata from each scan set normalized to provide for a ratio of expressionrelative to normal. Data from the microarray experiments was analyzedaccording to the algorithms described in U.S. application Ser. No.60/252,358, filed Nov. 20, 2000, by E. J. Moler, M. A. Boyle, and F. M.Randazzo, and entitled “Precision and accuracy in cDNA microarray data,”which application is specifically incorporated herein by reference.

[0180] The experiment was repeated, this time labeling the two probeswith the opposite color in order to perform the assay in both “colordirections.” Each experiment was sometimes repeated with two more slides(one in each color direction). The level fluorescence for each sequenceon the array expressed as a ratio of the geometric mean of 8 replicatespots/genes from the four arrays or 4 replicate spots/gene from 2 arraysor some other permutation. The data were normalized using the spikedpositive controls present in each duplicated area, and the precision ofthis normalization was included in the final determination of thesignificance of each differential. The fluorescent intensity of eachspot was also compared to the negative controls in each duplicated areato determine which spots have detected significant expression levels ineach sample.

[0181] A statistical analysis of the fluorescent intensities was appliedto each set of duplicate spots to assess the precision and significanceof each differential measurement, resulting in a p-value testing thenull hypothesis that there is no differential in the expression levelbetween the tumor and normal samples of each patient. During initialanalysis of the microarrays, the hypothesis was accepted if p>10⁻³, andthe differential ratio was set to 1.000 for those spots. All other spotshave a significant difference in expression between the tumor and normalsample. If the tumor sample has detectable expression and the normaldoes not, the ratio is truncated at 1000 since the value for expressionin the normal sample would be zero, and the ratio would not be amathematically useful value (e.g., infinity). If the normal sample hasdetectable expression and the tumor does not, the ratio is truncated to0.001, since the value for expression in the tumor sample would be zeroand the ratio would not be a mathematically useful value. These lattertwo situations are referred to herein as “on/off.” Database tables werepopulated using a 95% confidence level (p>0.05).

[0182] The difference in the expression level of TTK in the colon tumorcells relative to the matched normal colon cells was greater than orequal to 2 fold (“>=2×”) in 39% of the patients, greater than or equalto 2.5 fold in 36% of the patients, and greater than or equal to 5 foldin 27% of the patients examined.

[0183] Quantitative PCR of a number of normal tissues and tumor celllines, particularly colorectal carcinoma cell lines was used to analyzeexpression of TTK. Quantitative real-time PCR was performed by firstisolating RNA from cells using a Roche RNA Isolation kit according tomanufacturer's directions. One microgram of RNA was used to synthesize afirst-strand cDNA using MMLV reverse transcriptase (Ambion) using themanufacturers buffer and recommended concentrations of oligo dT,nucleotides, and Rnasin. This first-strand cDNA served as a template forquantitative real-time PCR using the Roche light-cycler as recommendedin the machine manual. TTK was amplified with the forward primerCGGAATCAAGTCTTCTAGCT (SEQ ID NO: 1) and reverse primerGGTTGCTCAAAAGTTGGTATG (SEQ ID NO:2) PCR product was quantified based onthe cycle at which the amplification entered the linear phase ofamplification in comparison to an internal standard and using thesoftware supplied by the manufacturer. Small differences in amounts ortotal template in the first-strand cDNA reaction were eliminated bynormalizing to amount of actin amplified in a separate quantitative PCRreaction using the forward primer 5′-CGGGAAATCGTGCGTGACATTAAG-3′ (SEQ IDNO:3) and the reverse primer: 5′-TGATCTCCTTCTGCATCCTGTCGG-3′ (SEQ IDNO:4). The results for TTK mRNA levels in normal tissues are shown inFIG. 1; the results for TTK mRNA levels in tumor cell lines are shown inFIG. 2. A brief description of the cell lines analyzed is provided inthe table below. Cell Line Tissue Source Cell Line Tissue SourceMDA-MB-231 Human breast; high Caco-2 Human colorectal metastaticpotential adenocarcinoma (micromets in lung; adenocarcinoma; pleuraleffusion MDA-MB-435 Human breast, high SW620 Human colorectal metastaticpotential adenocarcinoma; (macrometastases in from metastatic lung) site(lymph node) MCF-7 Human breast; non- LS174T High metastatic metastaticpotential human colorectal adenocarcinoma MDA-MB-468 Human breast; LOVOHuman colorectal adenocarcinoma adenocarcinoma; colon; from metastaticsite (colon) Alab Human breast, HT29 Human colorectal metastaticadenocarcinoma; colon SKOV3 Human ovarian SW480 Human colorectaladenocarcinoma adenocarcinoma; colon OVCAR3 Human ovarian HCT116 Humancolorectal adenocarcinoma carcinoma; colon KM12C Human colon; low Colo320DN Human colorectal metastatic potential adenocarcinoma; colon KM12L4Human colon; high T84 Human colorectal metastatic potential carcinoma;colon; (derived from from metastatic site Km12C) (lung) DU 145 Humanprostate; HCT15 Human colorectal carcinoma; from adenocarcinoma;metastatic site: brain colon HT1080 Human sarcoma cell CCD112 Humancolorectal line; adenocarcinoma, low metastatic potential HMVEC Primaryhuman DLD1 Human colon; microvascular colorectal endothelial cellsadenocarcinoma 184B5 normal breast 293 kidney epithelial epithelialcells; cells chemically trans- formed LNCAP prostate carcinoma; GRDP2primary prostate metastasis to left epithelium supraclavicular lymphU373MG glioblastoma cell IMR90 primary lung fibroblast WOCA primaryprostate PC3 prostate cancer; epithelium androgen receptor negative

[0184] TTK was expressed in normal cells (FIG. 1), with thymus andtestis identified as the normal tissues that most highly expressed thegene for TTK. Numerous cancer cells, however, displayed a significantlyelevated level of TTK expression (FIG. 2) as compared to most wild-typetissues.

Example 3 Hierarchical Clustering and Stratification of Colon CancersUsing Differential Expression Data

[0185] Differential expression patterns from Example 2 were analyzed byapplying hierarchical clustering methods to the data sets (see Eisen etal. (1998) PNAS 95:14863-14868). In short, hierarchical clusteringalgorithms are based on the average-linkage method of Sokal and Michener(Sokal, R R & Michener, C D (1958) Univ. Kans. Sci. Bull. 38,1409-1438), which was developed for clustering correlation matrixes. Theobject of this algorithm is to compute a dendrogram that assembles allelements into a single tree. For any set of n genes, an upper-diagonalsimilarity matrix is computed which contains similarity scores for allpairs of genes. The matrix is scanned to identify the highest value(representing a similar pair of genes). Using this technique, fourgroups of differential expression patterns were identified and assignedto clusters.

[0186] Application of hierarchical clustering to the data from Example 2revealed that IGF2 (insulin-like growth factor 2), TTK (serine,threonine, tyrosine kinase implicated in the cell cycle), MAPKAPK2(mitogen-activated protein (MAP) kinase-activated protein kinase), andMARCKS (myristoylated alanine-rich C kinase substrate, which is asubstrate of protein kinase C) are concurrently upregulated as detectedin 9 out of the 33 colon cancer patient samples examined. The data forthese experiments is presented in graphical form in FIGS. 3-6. Theconcurrent upregulation suggests that these genes are co-regulated andthat patients with an elevated serum level of IGF2 may be candidates fortreatment with inhibitors to TTK, MAPKAP kinase 2, MARCKS and/or IGF2.

Example 4 Antisense Regulation of TTK Expression

[0187] Additional functional information on TTK was generated usingantisense knockout technology. TTK expression in cancerous cells wasfurther analyzed to confirm the role and function of the gene product intumorgenesis, e.g., in promoting a metastatic phenotype.

[0188] A number of different oligonucleotides complementary to TTK mRNAwere designed as potential antisense oligonucleotides, and tested fortheir ability to suppress expression of TTK. The ability of eachdesigned antisense oligonucleotide to inhibit gene expression was testedthrough transfection into SW620 colon colorectal carcinoma cells. Foreach transfection mixture, a carrier molecule, preferably a lipitoid orcholesteroid, was prepared to a working concentration of 0.5 mM inwater, sonicated to yield a uniform solution, and filtered through a0.45 μm PVDF membrane. The antisense or control oligonucleotide was thenprepared to a working concentration of 100 μM in sterile Milliporewater. The oligonucleotide was further diluted in OptiMEM™ (Gibco/BRL),in a microfuge tube, to 2 μM, or approximately 20 μg oligo/ml ofOptiMEM™. In a separate microfuge tube, lipitoid or cholesteroid,typically in the amount of about 1.5-2 nmol lipitoid/pg antisenseoligonucleotide, was diluted into the same volume of OptiMEM™ used todilute the oligonucleotide. The diluted antisense oligonucleotide wasimmediately added to the diluted lipitoid and mixed by pipetting up anddown. Oligonucleotide was added to the cells to a final concentration of30 nM.

[0189] The level of target mRNA (TTK) in the transfected cells wasquantitated in the cancer cell lines using the Roche LightCycler™real-time PCR machine. Values for the target mRNA were normalized versusan internal control (e.g., beta-actin). For each 20 μl reaction,extracted RNA (generally 0.2-1 μg total) was placed into a sterile 0.5or 1.5 ml microcentrifuge tube, and water was added to a total volume of12.5 μl. To each tube was added 7.5 μl of a buffer/enzyme mixture,prepared by mixing (in the order listed) 2.5 μl H₂O, 2.0 μl 10×reactionbuffer, 10 μl oligo dT (20 pmol), 1.0 μl dNTP mix (10 mM each), 0.5 μlRNAsin® (20 u) (Ambion, Inc., Hialeah, Fla.), and 0.5 μl MMLV reversetranscriptase (50 u) (Ambion, Inc.). The contents were mixed bypipetting up and down, and the reaction mixture was incubated at 42° C.for 1 hour. The contents of each tube were centrifuged prior toamplification.

[0190] An amplification mixture was prepared by mixing in the followingorder: 1×PCR buffer II, 3 mM MgCl₂, 140 μM each dNTP, 0.175 pmol eacholigo, 1:50,000 dil of SYBR® Green, 0.25 mg/ml BSA, 1 unit Taqpolymerase, and H₂O to 20 μl. (PCR buffer II is available in10×concentration from Perkin-Elmer, Norwalk, Conn.). In 1×concentrationit contains 10 mM Tris pH 8.3 and 50 mM KCl. SYBR® Green (MolecularProbes, Eugene, Oreg.) is a dye which fluoresces when bound to doublestranded DNA. As double stranded PCR product is produced duringamplification, the fluorescence from SYBR® Green increases. To each 20μl aliquot of amplification mixture, 2 μl of template RT was added, andamplification was carried out according to standard protocols.

[0191] The following antisense oligonucleotides were shown toeffectively deplete TTK RNA in the transfection assays:

[0192] Oligo 79-5AS: GGGACTCTTCCAAATGGGCATGACT (SEQ ID NO:5)

[0193] Oligo 79-9AS: TCCAGTAACTCTTGCGTTCCCATGG (SEQ ID NO:6)

[0194] The reverse control of each of these antisense oligonucleotideswere synthesized, as were oligonucleotides with the identical sequenceof the antisense oligonucleotides in reverse orientation (ReverseControl):

[0195] Oligo 79-5RC: TCAGTACGGGTAAACCTTCTCAGGG (SEQ ID NO:7)

[0196] Oligo 79-9RC: GGTACCCTTGCGTTCTCAATGACCT (SEQ ID NO:8)

[0197] The antisense oligonucleotides were introduced into a test celland the effect upon TTK expression of the corresponding gene, as well asthe effect induction of the cancerous phenotype, was examined asdescribed below.

Example 5 Effect of TTK Expression on Proliferation

[0198] The effect of TTK on proliferation was assessed in metastaticbreast cancer cell lines (MDA-MB-231 (“231”)), SW620 colon colorectalcarcinoma cells, or 847 human immortal fibroblast cells. Transfectionwas carried out as described above in Example 4.

[0199] Cells were plated to approximately 60-80% confluency in 96-welldishes. Antisense or reverse control oligonucleotide was diluted to 2 μMin OptiMEM™ and added to OptiMEM™ into which the delivery vehicle,lipitoid 116-6 in the case of SW620 cells or 1:1 lipitoid 1:cholesteroid1 in the case of MDA-MB-231 cells, had been diluted. The oligo/ deliveryvehicle mixture was then further diluted into medium with serum on thecells. The final concentration of oligonucleotide for all experimentswas 300 nM, and the final ratio of oligo to delivery vehicle for allexperiments was 1.5 nmol lipitoid/μg oligonucleotide. Cells weretransfected overnight at 37° C. and the transfection mixture wasreplaced with fresh medium the next morning.

[0200] Transfection of the antisense oligonucleotides into both SW620colorectal carcinoma cells (FIG. 7) and 231 cells (FIG. 8) resulted in adecreased rate of proliferation compared to matched reverse control (RC)and oligonucleotides, but no inhibition of growth of 847 human immortalfibroblast cells (FIG. 11), suggesting possible tissue or transformationspecificity in the functional role for the TTK protein.

Example 6 Effect of TTK Expression on Colony Formation

[0201] The effect of TTK expression upon colony formation was tested ina soft agar assay. Soft agar assays were conducted by first establishinga bottom layer of 2 ml of 0.6% agar in media plated fresh within a fewhours of layering on the cells. The cell layer was formed on the bottomlayer by removing cells transfected as described above from plates using0.05% trypsin and washing twice in media. The cells were counted in aCoulter counter, and resuspended to 106 per ml in media. 10 μl aliquotsare placed with media in 96-well plates (to check counting with WST1),or diluted further for soft agar assay. 2000 cells are plated in 800 μl0.4% agar in duplicate wells above 0.6% agar bottom layer. After thecell layer agar solidifies, 2 ml of media is dribbled on top andantisense or reverse control oligo is added without delivery vehicles.Fresh media and oligos are added every 3-4 days. Colonies are formed in10 days to 3 weeks. Fields of colonies were counted by eye. Wst-1metabolism values can be used to compensate for small differences instarting cell number. Larger fields can be scanned for visual record ofdifferences.

[0202] As shown in FIG. 9, antisense oligonucleotides to TTK (79-9AS)led to decreased colony size and number compared to control reversecontrol oligonucleotides (79-9RC) or to control oligonucleotides(52-3AS: TAGGTCTTTGGCCGGTGATGGGTCG (SEQ ID NO:9) and 52-3RC:GCTGGGTAGTGGCCGGTTTCTGGAT (SEQ ID NO:10)). The 52-3 antisenseoligonucleotide is directed to the hD53 mRNA, and serves as a negativecontrol in the experiment.

Example 7 Induction of Cell Death Upon Depletion of TTK (“AntisenseKnockout”)

[0203] SW620 cells were transfected as described for proliferationassays. For cytotoxic effect in the presence of cisplatin (cis), thesame protocol was followed but cells were left in the presence of 2 μMdrug. Each day, cytotoxicity was monitored by measuring the amount ofLDH enzyme released in the medium due to membrane damage. The activityof LDH was measured using the Cytotoxicity Detection Kit from RocheMolecular Biochemicals. The data is provided as a ratio of LDH releasedin the medium vs. the total LDH present in the well at the same timepoint and treatment (rLDH/tLDH). A positive control using antisense andreverse control oligonucleotides for BCL2 (a known anti-apoptotic gene)shows that loss of message for BCL2 leads to an increase in cell deathcompared with treatment with the control oligonucleotide (backgroundcytotoxicity due to transfection).

[0204] The following antisense oligonucleotides were tested for theability to deplete the message levels of the gene corresponding to theindicated cluster. Oligo Name: AS or RC provides the name of the targetgene or name of the oligo, and whether the oligo is antisense (AS) or areverse control (RC). Oligo Name: Antisense (AS) or Reverse Control (RC)Oligo Sequence SEQ ID NO: Chir39-5:AS ACTCATCTGGCTGGGCTATGGTGGT SEQ IDNO:11 Chir39-5:RC TGGTGGTATCGGGTCGGTCTACTCA SEQ ID NO:12 Chir79-9:ASTCCAGTAACTCTTGCGTTCCCATGG SEQ ID NO:6 Chir79-9:RCGGTACCCTTGCGTTCTCAATGACCT SEQ ID NO:8

[0205] As shown in FIG. 12, Chiron 79-9 (TTK) antisense does notsensitize the cells to treatment by cisplatin at a detectable level, butleads to increased death compared to control oligo at day 3.

Example 8 Sample Assay for Agents that Modulate TTK Activity

[0206] This assay may be performed in microtitre plates. TTK waspurified as a 6×IIis tagged fusion protein using a baculovirusexpression system. Essentially 20 ul of 20 nM TTK (100 k Da) in TTKkinase buffer comprising 50 mM Hepes pH 7.4, 2mM MgCl₂, 10 mM MnCl₂, 1mM NaF, 50 mM NaCl, 1 mM DTT and 1 mg/ml BSA was added to 5 ul of acandidate agent diluted in 20% DMSO, 10 ul of a 2.8 uM solution of abiotinylated substrate peptide derived from cdc25, such asBiotin-SGSGSGLYRSPSMPENLNRPR-NH2 (SEQ ID NO:27) orBiotin-GGGGLYRSPSMPENLNRK-OH (SEQ ID NO:28) and 5 ul of 80 nM ³³P-γATPin a well of a microtitre plate. Samples were mixed, incubated for 2hours and each reaction is terminated using 20 ul of 0.5 M EDTA pH 8.0.50 ul of the sample is transferred to a 96 well flat bottom Streptavidincoated flash plate, and the sample is incubated with the plate for 1 hrat room temperature. The wells of the plate are washed four times with250 ul of calcium and magnesium-free phosphate buffered saline, andscintillation fluid is added to the sample. Activity of TTK was measuredby calculating the emission of ³³P, transferred by TTK from ³³P-γATP toa substrate peptide, by scintillation.

[0207] Agents modulating TTK activity can be identified by comparing theactivity of TTK in the presence of a candidate agent to the activity ofTTK in the absence of a candidate agent.

[0208] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe invention.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 38 <210> SEQ ID NO 1<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide<400> SEQUENCE: 1 cggaatcaag tcttctagct 20 <210> SEQ ID NO 2 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: synthesized oligonucleotide <400>SEQUENCE: 2 ggttgctcaa aagttggtat g 21 <210> SEQ ID NO 3 <211> LENGTH:24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthesized oligonucleotide <400> SEQUENCE: 3cgggaaatcg tgcgtgacat taag 24 <210> SEQ ID NO 4 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthesized oligonucleotide <400> SEQUENCE: 4 tgatctccttctgcatcctg tcgg 24 <210> SEQ ID NO 5 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthesized oligonucleotide <400> SEQUENCE: 5 gggactcttccaaatgggca tgact 25 <210> SEQ ID NO 6 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthesized oligonucleotide <400> SEQUENCE: 6 tccagtaactcttgcgttcc catgg 25 <210> SEQ ID NO 7 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthesized oligonucleotide <400> SEQUENCE: 7 tcagtacgggtaaaccttct caggg 25 <210> SEQ ID NO 8 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthesized oligonucleotide <400> SEQUENCE: 8 ggtacccttgcgttctcaat gacct 25 <210> SEQ ID NO 9 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthesized oligonucleotide <400> SEQUENCE: 9 taggtctttggccggtgatg ggtcg 25 <210> SEQ ID NO 10 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthesized oligonucleotide <400> SEQUENCE: 10 gctgggtagtggccggtttc tggat 25 <210> SEQ ID NO 11 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthesized oligonucleotide <400> SEQUENCE: 11 actcatctggctgggctatg gtggt 25 <210> SEQ ID NO 12 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: synthesized oligonucleotide <400> SEQUENCE: 12 tggtggtatcgggtcggtct actca 25 <210> SEQ ID NO 13 <211> LENGTH: 3866 <212> TYPE:DNA <213> ORGANISM: Homo sapien <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1026)...(3551) <221> NAME/KEY: misc_feature <222> LOCATION:(0)...(0) <223> OTHER INFORMATION: TTK <400> SEQUENCE: 13 ggaattcctttttttttttt tttgagatgg agtttcactc ttgttggcca ggctggagtg 60 caatggcacaatctcagctt actgcaacct ccgcctcccg ggttcaagcg attctcctgc 120 ctcagcctctcaagtagctg ggattacagg catgtgccac cacccctggc taactaattt 180 cttttctatttagtagagat ggggtttcac catgttggtc aggctggtct tgaactcctg 240 acctcaggtgatccacttgc cttggcctcc caaagtgcta ggattacagc cgtgaaactg 300 tgcctggctgattctttttt tgttgttgga tttttgaaac agggtctccc ttggtcgccc 360 aggctggagtgcagtggtgc gatcttggct cactataacc tccacctcct ggtttcaagt 420 gatcctcccactttagcctc ctgagtagct gtgattacag gcgtgcacca ccacacccgg 480 ctaatttttgtatttttatt agagacaggg tttcaccatg ttggccaggc tgttctcaaa 540 ctcctggactcaagggatcc gcctgcctcc acttcccaaa gtcccgagat tacaggtgtg 600 agtcaccatgcctgacctta taattcttaa gtcatttttt ctggtccatt tcttccttag 660 ggtcctcacaacaaatctgc attaggcggt acaataatcc ttaacttcat gattcacaaa 720 aggaagatgaagtgattcat gatttagaaa ggggaagtag taagcccact gcacactcct 780 ggatgatgatcctaaatcca gatacagtaa aaatggggta tgggaaggta gaatacaaaa 840 tttggtttaaattaattatc taaatatcta aaaacatttt tggatacatt gttgatgtga 900 atgtaagactgtacagactt cctagaaaac agtttgggtt ccatcttttc atttccccag 960 tgcagttttctgtagaaatg gaatccgagg atttaagtgg cagagaattg acaattgatt 1020 ccata atgaac aaa gtg aga gac att aaa aat aag ttt aaa aat gaa gac 1070 Met Asn LysVal Arg Asp Ile Lys Asn Lys Phe Lys Asn Glu Asp 1 5 10 15 ctt act gatgaa cta agc ttg aat aaa att tct gct gat act aca gat 1118 Leu Thr Asp GluLeu Ser Leu Asn Lys Ile Ser Ala Asp Thr Thr Asp 20 25 30 aac tcg gga actgtt aac caa att atg atg atg gca aac aac cca gag 1166 Asn Ser Gly Thr ValAsn Gln Ile Met Met Met Ala Asn Asn Pro Glu 35 40 45 gac tgg ttg agt ttgttg ctc aaa cta gag aaa aac agt gtt ccg cta 1214 Asp Trp Leu Ser Leu LeuLeu Lys Leu Glu Lys Asn Ser Val Pro Leu 50 55 60 agt gat gct ctt tta aataaa ttg att ggt cgt tac agt caa gca att 1262 Ser Asp Ala Leu Leu Asn LysLeu Ile Gly Arg Tyr Ser Gln Ala Ile 65 70 75 gaa gcg ctt ccc cca gat aaatat ggc caa aat gag agt ttt gct aga 1310 Glu Ala Leu Pro Pro Asp Lys TyrGly Gln Asn Glu Ser Phe Ala Arg 80 85 90 95 att caa gtg aga ttt gct gaatta aaa gct att caa gag cca gat gat 1358 Ile Gln Val Arg Phe Ala Glu LeuLys Ala Ile Gln Glu Pro Asp Asp 100 105 110 gca cgt gac tac ttt caa atggcc aga gca aac tgc aag aaa ttt gct 1406 Ala Arg Asp Tyr Phe Gln Met AlaArg Ala Asn Cys Lys Lys Phe Ala 115 120 125 ttt gtt cat ata tct ttt gcacaa ttt gaa ctg tca caa ggt aat gtc 1454 Phe Val His Ile Ser Phe Ala GlnPhe Glu Leu Ser Gln Gly Asn Val 130 135 140 aaa aaa agt aaa caa ctt cttcaa aaa gct gta gaa cgt gga gca gta 1502 Lys Lys Ser Lys Gln Leu Leu GlnLys Ala Val Glu Arg Gly Ala Val 145 150 155 cca cta gaa atg ctg gaa attgcc ctg cgg aat tta aac ctc caa aaa 1550 Pro Leu Glu Met Leu Glu Ile AlaLeu Arg Asn Leu Asn Leu Gln Lys 160 165 170 175 aag cag ctg ctt tca gaggag gaa aag aag aat tta tca gca tct acg 1598 Lys Gln Leu Leu Ser Glu GluGlu Lys Lys Asn Leu Ser Ala Ser Thr 180 185 190 gta tta act gcc caa gaatca ttt tcc ggt tca ctt ggg cat tta cag 1646 Val Leu Thr Ala Gln Glu SerPhe Ser Gly Ser Leu Gly His Leu Gln 195 200 205 aat agg aac aac agt tgtgat tcc aga gga cag act act aaa gcc agg 1694 Asn Arg Asn Asn Ser Cys AspSer Arg Gly Gln Thr Thr Lys Ala Arg 210 215 220 ttt tta tat gga gag aacatg cca cca caa gat gca gaa ata ggt tac 1742 Phe Leu Tyr Gly Glu Asn MetPro Pro Gln Asp Ala Glu Ile Gly Tyr 225 230 235 cgg aat tca ttg aga caaact aac aaa act aaa cag tca tgc cca ttt 1790 Arg Asn Ser Leu Arg Gln ThrAsn Lys Thr Lys Gln Ser Cys Pro Phe 240 245 250 255 gga aga gtc cca gttaac ctt cta aat agc cca gat tgt gat gtg aag 1838 Gly Arg Val Pro Val AsnLeu Leu Asn Ser Pro Asp Cys Asp Val Lys 260 265 270 aca gat gat tca gttgta cct tgt ttt atg aaa aga caa acc tct aga 1886 Thr Asp Asp Ser Val ValPro Cys Phe Met Lys Arg Gln Thr Ser Arg 275 280 285 tca gaa tgc cga gatttg gtt gtg cct gga tct aaa cca agt gga aat 1934 Ser Glu Cys Arg Asp LeuVal Val Pro Gly Ser Lys Pro Ser Gly Asn 290 295 300 gat tcc tgt gaa ttaaga aat tta aag tct gtt caa aat agt cat ttc 1982 Asp Ser Cys Glu Leu ArgAsn Leu Lys Ser Val Gln Asn Ser His Phe 305 310 315 aag gaa cct ctg gtgtca gat gaa aag agt tct gaa ctt att att act 2030 Lys Glu Pro Leu Val SerAsp Glu Lys Ser Ser Glu Leu Ile Ile Thr 320 325 330 335 gat tca ata accctg aag aat aaa acg gaa tca agt ctt cta gct aaa 2078 Asp Ser Ile Thr LeuLys Asn Lys Thr Glu Ser Ser Leu Leu Ala Lys 340 345 350 tta gaa gaa actaaa gag tat caa gaa cca gag gtt cca gag agt aac 2126 Leu Glu Glu Thr LysGlu Tyr Gln Glu Pro Glu Val Pro Glu Ser Asn 355 360 365 cag aaa cag tggcaa gct aag aga aag tca gag tgt att aac cag aat 2174 Gln Lys Gln Trp GlnAla Lys Arg Lys Ser Glu Cys Ile Asn Gln Asn 370 375 380 cct gct gca tcttca aat cac tgg cag att ccg gag tta gcc cga aaa 2222 Pro Ala Ala Ser SerAsn His Trp Gln Ile Pro Glu Leu Ala Arg Lys 385 390 395 gtt aat aca gagcag aaa cat acc act ttt gag caa cct gtc ttt tca 2270 Val Asn Thr Glu GlnLys His Thr Thr Phe Glu Gln Pro Val Phe Ser 400 405 410 415 gtt tca aaacag tca cca cca ata tca aca tct aaa tgg ttt gac cca 2318 Val Ser Lys GlnSer Pro Pro Ile Ser Thr Ser Lys Trp Phe Asp Pro 420 425 430 aaa tct atttgt aag aca cca agc agc aat acc ttg gat gat tac atg 2366 Lys Ser Ile CysLys Thr Pro Ser Ser Asn Thr Leu Asp Asp Tyr Met 435 440 445 agc tgt tttaga act cca gtt gta aag aat gac ttt cca cct gct tgt 2414 Ser Cys Phe ArgThr Pro Val Val Lys Asn Asp Phe Pro Pro Ala Cys 450 455 460 cag ttg tcaaca cct tat ggc caa cct gcc tgt ttc cag cag caa cag 2462 Gln Leu Ser ThrPro Tyr Gly Gln Pro Ala Cys Phe Gln Gln Gln Gln 465 470 475 cat caa atactt gcc act cca ctt caa aat tta cag gtt tta gca tct 2510 His Gln Ile LeuAla Thr Pro Leu Gln Asn Leu Gln Val Leu Ala Ser 480 485 490 495 tct tcagca aat gaa tgc att tcg gtt aaa gga aga att tat tcc ata 2558 Ser Ser AlaAsn Glu Cys Ile Ser Val Lys Gly Arg Ile Tyr Ser Ile 500 505 510 tta aagcag ata gga agt gga ggt tca agc aag gta ttt cag gtg tta 2606 Leu Lys GlnIle Gly Ser Gly Gly Ser Ser Lys Val Phe Gln Val Leu 515 520 525 aat gaaaag aaa cag ata tat gct ata aaa tat gtg aac tta gaa gaa 2654 Asn Glu LysLys Gln Ile Tyr Ala Ile Lys Tyr Val Asn Leu Glu Glu 530 535 540 gca gataac caa act ctt gat agt tac cgg aac gaa ata gct tat ttg 2702 Ala Asp AsnGln Thr Leu Asp Ser Tyr Arg Asn Glu Ile Ala Tyr Leu 545 550 555 aat aaacta caa caa cac agt gat aag atc atc cga ctt tat gat tat 2750 Asn Lys LeuGln Gln His Ser Asp Lys Ile Ile Arg Leu Tyr Asp Tyr 560 565 570 575 gaaatc acg gac cag tac atc tac atg gta atg gag tgt gga aat att 2798 Glu IleThr Asp Gln Tyr Ile Tyr Met Val Met Glu Cys Gly Asn Ile 580 585 590 gatctt aat agt tgg ctt aaa aag aaa aaa tcc att gat cca tgg gaa 2846 Asp LeuAsn Ser Trp Leu Lys Lys Lys Lys Ser Ile Asp Pro Trp Glu 595 600 605 cgcaag agt tac tgg aaa aat atg tta gag gca gtt cac aca atc cat 2894 Arg LysSer Tyr Trp Lys Asn Met Leu Glu Ala Val His Thr Ile His 610 615 620 caacat ggc att gtt cac agt gat ctt aaa cca gct aac ttt ctg ata 2942 Gln HisGly Ile Val His Ser Asp Leu Lys Pro Ala Asn Phe Leu Ile 625 630 635 gttgat gga atg cta aag cta att gat ttt ggg att gca aac caa atg 2990 Val AspGly Met Leu Lys Leu Ile Asp Phe Gly Ile Ala Asn Gln Met 640 645 650 655caa cca gat aca aca agt gtt gtt aaa gat tct cag gtt ggc aca gtt 3038 GlnPro Asp Thr Thr Ser Val Val Lys Asp Ser Gln Val Gly Thr Val 660 665 670aat tat atg cca cca gaa gca atc aaa gat atg tct tcc tcc aga gag 3086 AsnTyr Met Pro Pro Glu Ala Ile Lys Asp Met Ser Ser Ser Arg Glu 675 680 685aat ggg aaa tct aag tca aag ata agc ccc aaa agt gat gtt tgg tcc 3134 AsnGly Lys Ser Lys Ser Lys Ile Ser Pro Lys Ser Asp Val Trp Ser 690 695 700tta gga tgt att ttg tac tat atg act tac ggg aaa aca cca ttt cag 3182 LeuGly Cys Ile Leu Tyr Tyr Met Thr Tyr Gly Lys Thr Pro Phe Gln 705 710 715cag ata att aat cag att tct aaa tta cat gcc ata att gat cct aat 3230 GlnIle Ile Asn Gln Ile Ser Lys Leu His Ala Ile Ile Asp Pro Asn 720 725 730735 cat gaa att gaa ttt ccc gat att cca gag aaa gat ctt caa gat gtg 3278His Glu Ile Glu Phe Pro Asp Ile Pro Glu Lys Asp Leu Gln Asp Val 740 745750 tta aag tgt tgt tta aaa agg gac cca aaa cag agg ata tcc att cct 3326Leu Lys Cys Cys Leu Lys Arg Asp Pro Lys Gln Arg Ile Ser Ile Pro 755 760765 gag ctc ctg gct cat cca tat gtt caa att caa act cat cca gtt aac 3374Glu Leu Leu Ala His Pro Tyr Val Gln Ile Gln Thr His Pro Val Asn 770 775780 caa atg gcc aag gga acc act gaa gaa atg aaa tat gtt ctg ggc caa 3422Gln Met Ala Lys Gly Thr Thr Glu Glu Met Lys Tyr Val Leu Gly Gln 785 790795 ctt gtt ggt ctg aat tct cct aac tcc att ttg aaa gct gct aaa act 3470Leu Val Gly Leu Asn Ser Pro Asn Ser Ile Leu Lys Ala Ala Lys Thr 800 805810 815 tta tat gaa cac tat agt ggt ggt gaa agt cat aat tct tca tcc tcc3518 Leu Tyr Glu His Tyr Ser Gly Gly Glu Ser His Asn Ser Ser Ser Ser 820825 830 aag act ttt gaa aaa aaa agg gga aaa aaa tga tttgcagttattcgtaatgt 3571 Lys Thr Phe Glu Lys Lys Arg Gly Lys Lys * 835 840cagataggag gtataaaata tattggactg ttatactctt gaatccctgt ggaaatctac 3631atttgaagac aacatcactc tgaagtgtta tcagcaaaaa aaattcagtg agattatctt 3691taaaagaaaa ctgtaaaaat agcaaccact tatggcactg tatatattgt agacttgttt 3751tctctgtttt atgctcttgt gtaatctact tgacatcatt ttactcttgg aatagtgggt 3811ggatagcaag tatattctaa aaaactttgt aaataaagtt ttgtggctaa aatga 3866 <210>SEQ ID NO 14 <211> LENGTH: 841 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 14 Met Asn Lys Val Arg Asp Ile Lys Asn Lys PheLys Asn Glu Asp Leu 1 5 10 15 Thr Asp Glu Leu Ser Leu Asn Lys Ile SerAla Asp Thr Thr Asp Asn 20 25 30 Ser Gly Thr Val Asn Gln Ile Met Met MetAla Asn Asn Pro Glu Asp 35 40 45 Trp Leu Ser Leu Leu Leu Lys Leu Glu LysAsn Ser Val Pro Leu Ser 50 55 60 Asp Ala Leu Leu Asn Lys Leu Ile Gly ArgTyr Ser Gln Ala Ile Glu 65 70 75 80 Ala Leu Pro Pro Asp Lys Tyr Gly GlnAsn Glu Ser Phe Ala Arg Ile 85 90 95 Gln Val Arg Phe Ala Glu Leu Lys AlaIle Gln Glu Pro Asp Asp Ala 100 105 110 Arg Asp Tyr Phe Gln Met Ala ArgAla Asn Cys Lys Lys Phe Ala Phe 115 120 125 Val His Ile Ser Phe Ala GlnPhe Glu Leu Ser Gln Gly Asn Val Lys 130 135 140 Lys Ser Lys Gln Leu LeuGln Lys Ala Val Glu Arg Gly Ala Val Pro 145 150 155 160 Leu Glu Met LeuGlu Ile Ala Leu Arg Asn Leu Asn Leu Gln Lys Lys 165 170 175 Gln Leu LeuSer Glu Glu Glu Lys Lys Asn Leu Ser Ala Ser Thr Val 180 185 190 Leu ThrAla Gln Glu Ser Phe Ser Gly Ser Leu Gly His Leu Gln Asn 195 200 205 ArgAsn Asn Ser Cys Asp Ser Arg Gly Gln Thr Thr Lys Ala Arg Phe 210 215 220Leu Tyr Gly Glu Asn Met Pro Pro Gln Asp Ala Glu Ile Gly Tyr Arg 225 230235 240 Asn Ser Leu Arg Gln Thr Asn Lys Thr Lys Gln Ser Cys Pro Phe Gly245 250 255 Arg Val Pro Val Asn Leu Leu Asn Ser Pro Asp Cys Asp Val LysThr 260 265 270 Asp Asp Ser Val Val Pro Cys Phe Met Lys Arg Gln Thr SerArg Ser 275 280 285 Glu Cys Arg Asp Leu Val Val Pro Gly Ser Lys Pro SerGly Asn Asp 290 295 300 Ser Cys Glu Leu Arg Asn Leu Lys Ser Val Gln AsnSer His Phe Lys 305 310 315 320 Glu Pro Leu Val Ser Asp Glu Lys Ser SerGlu Leu Ile Ile Thr Asp 325 330 335 Ser Ile Thr Leu Lys Asn Lys Thr GluSer Ser Leu Leu Ala Lys Leu 340 345 350 Glu Glu Thr Lys Glu Tyr Gln GluPro Glu Val Pro Glu Ser Asn Gln 355 360 365 Lys Gln Trp Gln Ala Lys ArgLys Ser Glu Cys Ile Asn Gln Asn Pro 370 375 380 Ala Ala Ser Ser Asn HisTrp Gln Ile Pro Glu Leu Ala Arg Lys Val 385 390 395 400 Asn Thr Glu GlnLys His Thr Thr Phe Glu Gln Pro Val Phe Ser Val 405 410 415 Ser Lys GlnSer Pro Pro Ile Ser Thr Ser Lys Trp Phe Asp Pro Lys 420 425 430 Ser IleCys Lys Thr Pro Ser Ser Asn Thr Leu Asp Asp Tyr Met Ser 435 440 445 CysPhe Arg Thr Pro Val Val Lys Asn Asp Phe Pro Pro Ala Cys Gln 450 455 460Leu Ser Thr Pro Tyr Gly Gln Pro Ala Cys Phe Gln Gln Gln Gln His 465 470475 480 Gln Ile Leu Ala Thr Pro Leu Gln Asn Leu Gln Val Leu Ala Ser Ser485 490 495 Ser Ala Asn Glu Cys Ile Ser Val Lys Gly Arg Ile Tyr Ser IleLeu 500 505 510 Lys Gln Ile Gly Ser Gly Gly Ser Ser Lys Val Phe Gln ValLeu Asn 515 520 525 Glu Lys Lys Gln Ile Tyr Ala Ile Lys Tyr Val Asn LeuGlu Glu Ala 530 535 540 Asp Asn Gln Thr Leu Asp Ser Tyr Arg Asn Glu IleAla Tyr Leu Asn 545 550 555 560 Lys Leu Gln Gln His Ser Asp Lys Ile IleArg Leu Tyr Asp Tyr Glu 565 570 575 Ile Thr Asp Gln Tyr Ile Tyr Met ValMet Glu Cys Gly Asn Ile Asp 580 585 590 Leu Asn Ser Trp Leu Lys Lys LysLys Ser Ile Asp Pro Trp Glu Arg 595 600 605 Lys Ser Tyr Trp Lys Asn MetLeu Glu Ala Val His Thr Ile His Gln 610 615 620 His Gly Ile Val His SerAsp Leu Lys Pro Ala Asn Phe Leu Ile Val 625 630 635 640 Asp Gly Met LeuLys Leu Ile Asp Phe Gly Ile Ala Asn Gln Met Gln 645 650 655 Pro Asp ThrThr Ser Val Val Lys Asp Ser Gln Val Gly Thr Val Asn 660 665 670 Tyr MetPro Pro Glu Ala Ile Lys Asp Met Ser Ser Ser Arg Glu Asn 675 680 685 GlyLys Ser Lys Ser Lys Ile Ser Pro Lys Ser Asp Val Trp Ser Leu 690 695 700Gly Cys Ile Leu Tyr Tyr Met Thr Tyr Gly Lys Thr Pro Phe Gln Gln 705 710715 720 Ile Ile Asn Gln Ile Ser Lys Leu His Ala Ile Ile Asp Pro Asn His725 730 735 Glu Ile Glu Phe Pro Asp Ile Pro Glu Lys Asp Leu Gln Asp ValLeu 740 745 750 Lys Cys Cys Leu Lys Arg Asp Pro Lys Gln Arg Ile Ser IlePro Glu 755 760 765 Leu Leu Ala His Pro Tyr Val Gln Ile Gln Thr His ProVal Asn Gln 770 775 780 Met Ala Lys Gly Thr Thr Glu Glu Met Lys Tyr ValLeu Gly Gln Leu 785 790 795 800 Val Gly Leu Asn Ser Pro Asn Ser Ile LeuLys Ala Ala Lys Thr Leu 805 810 815 Tyr Glu His Tyr Ser Gly Gly Glu SerHis Asn Ser Ser Ser Ser Lys 820 825 830 Thr Phe Glu Lys Lys Arg Gly LysLys 835 840 <210> SEQ ID NO 15 <211> LENGTH: 2735 <212> TYPE: DNA <213>ORGANISM: Saccharomyces cerevisiae <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (34)...(2499) <400> SEQUENCE: 15 agaaaagata gtgttacacaacatcaacta aaa atg gaa aat att aca caa ccc 54 Met Glu Asn Ile Thr GlnPro 1 5 aca cag caa tcc acg cag gct act caa agg ttt ttg att gag aag ttt102 Thr Gln Gln Ser Thr Gln Ala Thr Gln Arg Phe Leu Ile Glu Lys Phe 1015 20 tct caa gaa cag atc ggc gaa aac att gtg tgc agg gtc att tgt acc150 Ser Gln Glu Gln Ile Gly Glu Asn Ile Val Cys Arg Val Ile Cys Thr 2530 35 acg ggt caa att ccc atc cga gat ttg tca gct gat att tca caa gtg198 Thr Gly Gln Ile Pro Ile Arg Asp Leu Ser Ala Asp Ile Ser Gln Val 4045 50 55 ctt aag gaa aaa cga tcc ata aag aaa gtt tgg aca ttt ggt aga aac246 Leu Lys Glu Lys Arg Ser Ile Lys Lys Val Trp Thr Phe Gly Arg Asn 6065 70 cca gcc tgt gac tat cat tta gga aac att tca aga ctg tca aat aag294 Pro Ala Cys Asp Tyr His Leu Gly Asn Ile Ser Arg Leu Ser Asn Lys 7580 85 cat ttc caa ata cta cta gga gaa gac ggt aac ctt tta ttg aat gac342 His Phe Gln Ile Leu Leu Gly Glu Asp Gly Asn Leu Leu Leu Asn Asp 9095 100 att tcc act aat ggg acc tgg tta aat ggg caa aaa gtc gag aag aac390 Ile Ser Thr Asn Gly Thr Trp Leu Asn Gly Gln Lys Val Glu Lys Asn 105110 115 agc aat cag tta ctg tct caa ggt gat gaa ata acc gtt ggt gta ggc438 Ser Asn Gln Leu Leu Ser Gln Gly Asp Glu Ile Thr Val Gly Val Gly 120125 130 135 gtg gaa tca gat att tta tct ctg gtc att ttc ata aac gac aaattt 486 Val Glu Ser Asp Ile Leu Ser Leu Val Ile Phe Ile Asn Asp Lys Phe140 145 150 aag cag tgc ctc gag cag aac aaa gtt gat cgc ata aga tct aacctg 534 Lys Gln Cys Leu Glu Gln Asn Lys Val Asp Arg Ile Arg Ser Asn Leu155 160 165 aaa aat acc tct aaa ata gct tct cct ggt ctt aca tca tct actgca 582 Lys Asn Thr Ser Lys Ile Ala Ser Pro Gly Leu Thr Ser Ser Thr Ala170 175 180 tca tca atg gtg gcc aac aag act ggt att ttt aag gat ttt tcgatt 630 Ser Ser Met Val Ala Asn Lys Thr Gly Ile Phe Lys Asp Phe Ser Ile185 190 195 att gac gaa gtg gtg ggc cag ggt gca ttt gcc aca gta aag aaagcc 678 Ile Asp Glu Val Val Gly Gln Gly Ala Phe Ala Thr Val Lys Lys Ala200 205 210 215 att gaa aga act act ggg aaa aca ttc gcg gtg aag att ataagt aaa 726 Ile Glu Arg Thr Thr Gly Lys Thr Phe Ala Val Lys Ile Ile SerLys 220 225 230 cgc aaa gta ata ggc aat atg gat ggt gtg aca aga gag ttagaa gta 774 Arg Lys Val Ile Gly Asn Met Asp Gly Val Thr Arg Glu Leu GluVal 235 240 245 ttg caa aag ctc aat cat cca agg ata gta cga ttg aaa ggattt tat 822 Leu Gln Lys Leu Asn His Pro Arg Ile Val Arg Leu Lys Gly PheTyr 250 255 260 gaa gat act gag agt tat tat atg gtg atg gag ttc gtt tctggt ggt 870 Glu Asp Thr Glu Ser Tyr Tyr Met Val Met Glu Phe Val Ser GlyGly 265 270 275 gac tta atg gat ttt gtt gct gct cat ggt gcg gtt gga gaagat gct 918 Asp Leu Met Asp Phe Val Ala Ala His Gly Ala Val Gly Glu AspAla 280 285 290 295 ggg agg gag ata tcc agg cag ata ctc aca gca ata aaatac att cac 966 Gly Arg Glu Ile Ser Arg Gln Ile Leu Thr Ala Ile Lys TyrIle His 300 305 310 tct atg ggc atc agc cat cgt gac cta aag ccc gat aatatt ctt att 1014 Ser Met Gly Ile Ser His Arg Asp Leu Lys Pro Asp Asn IleLeu Ile 315 320 325 gaa caa gac gat cct gta ttg gta aag ata acc gac tttggt ctg gca 1062 Glu Gln Asp Asp Pro Val Leu Val Lys Ile Thr Asp Phe GlyLeu Ala 330 335 340 aaa gta caa gga aat ggg tct ttt atg aaa acc ttc tgtggc act ttg 1110 Lys Val Gln Gly Asn Gly Ser Phe Met Lys Thr Phe Cys GlyThr Leu 345 350 355 gca tat gtg gca cct gaa gtc atc aga ggt aaa gat acatcc gta tct 1158 Ala Tyr Val Ala Pro Glu Val Ile Arg Gly Lys Asp Thr SerVal Ser 360 365 370 375 cct gat gaa tac gaa gaa agg aat gag tac tct tcgtta gtg gat atg 1206 Pro Asp Glu Tyr Glu Glu Arg Asn Glu Tyr Ser Ser LeuVal Asp Met 380 385 390 tgg tca atg gga tgt ctt gtg tat gtt atc cta acgggc cac tta cct 1254 Trp Ser Met Gly Cys Leu Val Tyr Val Ile Leu Thr GlyHis Leu Pro 395 400 405 ttt agt ggt agc aca cag gac caa tta tat aaa cagatt gga aga ggc 1302 Phe Ser Gly Ser Thr Gln Asp Gln Leu Tyr Lys Gln IleGly Arg Gly 410 415 420 tca tat cat gaa ggg ccc ctc aaa gat ttc cgg atatct gaa gaa gca 1350 Ser Tyr His Glu Gly Pro Leu Lys Asp Phe Arg Ile SerGlu Glu Ala 425 430 435 aga gat ttc ata gat tca ttg tta cag gtg gat ccaaat aat agg tcg 1398 Arg Asp Phe Ile Asp Ser Leu Leu Gln Val Asp Pro AsnAsn Arg Ser 440 445 450 455 aca gct gca aaa gcc ttg aat cat ccc tgg atcaag atg agt cca ttg 1446 Thr Ala Ala Lys Ala Leu Asn His Pro Trp Ile LysMet Ser Pro Leu 460 465 470 ggc tca caa tca tat ggt gat ttt tca caa atatcc tta tca caa tcg 1494 Gly Ser Gln Ser Tyr Gly Asp Phe Ser Gln Ile SerLeu Ser Gln Ser 475 480 485 ttg tcg cag cag aaa tta tta gaa aat atg gacgat gct caa tac gaa 1542 Leu Ser Gln Gln Lys Leu Leu Glu Asn Met Asp AspAla Gln Tyr Glu 490 495 500 ttt gtc aaa gcg caa agg aaa tta caa atg gagcaa caa ctt caa gaa 1590 Phe Val Lys Ala Gln Arg Lys Leu Gln Met Glu GlnGln Leu Gln Glu 505 510 515 cag gat cag gaa gac caa gat gga aaa att caagga ttt aaa ata ccc 1638 Gln Asp Gln Glu Asp Gln Asp Gly Lys Ile Gln GlyPhe Lys Ile Pro 520 525 530 535 gca cac gcc cct att cga tat aca cag cccaaa agc att gaa gca gaa 1686 Ala His Ala Pro Ile Arg Tyr Thr Gln Pro LysSer Ile Glu Ala Glu 540 545 550 act aga gaa caa aaa ctt tta cat tcc aataat act gag aat gtc aag 1734 Thr Arg Glu Gln Lys Leu Leu His Ser Asn AsnThr Glu Asn Val Lys 555 560 565 agc tca aag aaa aag ggt aat ggt agg ttttta act tta aaa cca ttg 1782 Ser Ser Lys Lys Lys Gly Asn Gly Arg Phe LeuThr Leu Lys Pro Leu 570 575 580 cct gac agc att att caa gaa agc ctg gagatt cag caa ggt gtg aat 1830 Pro Asp Ser Ile Ile Gln Glu Ser Leu Glu IleGln Gln Gly Val Asn 585 590 595 cca ttt ttc att ggt aga tcc gag gat tgcaat tgt aaa att gaa gac 1878 Pro Phe Phe Ile Gly Arg Ser Glu Asp Cys AsnCys Lys Ile Glu Asp 600 605 610 615 aat agg ttg tct cga gtt cat tgc ttcatt ttc aaa aag agg cat gct 1926 Asn Arg Leu Ser Arg Val His Cys Phe IlePhe Lys Lys Arg His Ala 620 625 630 gta ggc aaa agc atg tat gaa tct ccggca caa ggt tta gat gat att 1974 Val Gly Lys Ser Met Tyr Glu Ser Pro AlaGln Gly Leu Asp Asp Ile 635 640 645 tgg tat tgc cac acc gga act aac gtgagc tat tta aat aat aac cgc 2022 Trp Tyr Cys His Thr Gly Thr Asn Val SerTyr Leu Asn Asn Asn Arg 650 655 660 atg ata cag ggt acg aaa ttc ctt ttacaa gac gga gat gaa atc aag 2070 Met Ile Gln Gly Thr Lys Phe Leu Leu GlnAsp Gly Asp Glu Ile Lys 665 670 675 atc att tgg gat aaa aac aat aaa tttgtc att ggc ttt aaa gtg gaa 2118 Ile Ile Trp Asp Lys Asn Asn Lys Phe ValIle Gly Phe Lys Val Glu 680 685 690 695 att aac gat act aca ggt ctg tttaac gag gga tta ggt atg tta caa 2166 Ile Asn Asp Thr Thr Gly Leu Phe AsnGlu Gly Leu Gly Met Leu Gln 700 705 710 gaa caa aga gta gta ctt aag caaaca gcc gaa gaa aaa gat ttg gtg 2214 Glu Gln Arg Val Val Leu Lys Gln ThrAla Glu Glu Lys Asp Leu Val 715 720 725 aaa aag tta acc cag atg atg gcagct caa cgt gca aat caa ccc tcg 2262 Lys Lys Leu Thr Gln Met Met Ala AlaGln Arg Ala Asn Gln Pro Ser 730 735 740 gct tct tct tca tca atg tcg gctaag aag ccg cca gtt agc gat aca 2310 Ala Ser Ser Ser Ser Met Ser Ala LysLys Pro Pro Val Ser Asp Thr 745 750 755 aat aat aac ggc aat aat tcg gtacta aac gac ttg gta gag tca ccg 2358 Asn Asn Asn Gly Asn Asn Ser Val LeuAsn Asp Leu Val Glu Ser Pro 760 765 770 775 att aat gcg aat acg ggg aacatt ttg aag aga ata cat tcg gta agt 2406 Ile Asn Ala Asn Thr Gly Asn IleLeu Lys Arg Ile His Ser Val Ser 780 785 790 tta tcg caa tca caa att gatcct agt aag aag gtt aaa agg gca aaa 2454 Leu Ser Gln Ser Gln Ile Asp ProSer Lys Lys Val Lys Arg Ala Lys 795 800 805 ttg gac caa acc tca aaa ggcccc gag aat ttg caa ttt tcg taa 2499 Leu Asp Gln Thr Ser Lys Gly Pro GluAsn Leu Gln Phe Ser * 810 815 820 ccaaggacaa atacccatag aaaatgctgcccctttttaa gagagaagat ggtagatacc 2559 aatactcaga attcccagta caaagaaccaatatcggagt caataaacag tatgatgaac 2619 ttgctttcgc aaataaaaga tatcactcagaagcacccag taataaagga tgcagatagc 2679 tcgagatttg gtaaggttga gtttagggacttttatgacg aagtttcacg gaattc 2735 <210> SEQ ID NO 16 <211> LENGTH: 821<212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE:16 Met Glu Asn Ile Thr Gln Pro Thr Gln Gln Ser Thr Gln Ala Thr Gln 1 510 15 Arg Phe Leu Ile Glu Lys Phe Ser Gln Glu Gln Ile Gly Glu Asn Ile 2025 30 Val Cys Arg Val Ile Cys Thr Thr Gly Gln Ile Pro Ile Arg Asp Leu 3540 45 Ser Ala Asp Ile Ser Gln Val Leu Lys Glu Lys Arg Ser Ile Lys Lys 5055 60 Val Trp Thr Phe Gly Arg Asn Pro Ala Cys Asp Tyr His Leu Gly Asn 6570 75 80 Ile Ser Arg Leu Ser Asn Lys His Phe Gln Ile Leu Leu Gly Glu Asp85 90 95 Gly Asn Leu Leu Leu Asn Asp Ile Ser Thr Asn Gly Thr Trp Leu Asn100 105 110 Gly Gln Lys Val Glu Lys Asn Ser Asn Gln Leu Leu Ser Gln GlyAsp 115 120 125 Glu Ile Thr Val Gly Val Gly Val Glu Ser Asp Ile Leu SerLeu Val 130 135 140 Ile Phe Ile Asn Asp Lys Phe Lys Gln Cys Leu Glu GlnAsn Lys Val 145 150 155 160 Asp Arg Ile Arg Ser Asn Leu Lys Asn Thr SerLys Ile Ala Ser Pro 165 170 175 Gly Leu Thr Ser Ser Thr Ala Ser Ser MetVal Ala Asn Lys Thr Gly 180 185 190 Ile Phe Lys Asp Phe Ser Ile Ile AspGlu Val Val Gly Gln Gly Ala 195 200 205 Phe Ala Thr Val Lys Lys Ala IleGlu Arg Thr Thr Gly Lys Thr Phe 210 215 220 Ala Val Lys Ile Ile Ser LysArg Lys Val Ile Gly Asn Met Asp Gly 225 230 235 240 Val Thr Arg Glu LeuGlu Val Leu Gln Lys Leu Asn His Pro Arg Ile 245 250 255 Val Arg Leu LysGly Phe Tyr Glu Asp Thr Glu Ser Tyr Tyr Met Val 260 265 270 Met Glu PheVal Ser Gly Gly Asp Leu Met Asp Phe Val Ala Ala His 275 280 285 Gly AlaVal Gly Glu Asp Ala Gly Arg Glu Ile Ser Arg Gln Ile Leu 290 295 300 ThrAla Ile Lys Tyr Ile His Ser Met Gly Ile Ser His Arg Asp Leu 305 310 315320 Lys Pro Asp Asn Ile Leu Ile Glu Gln Asp Asp Pro Val Leu Val Lys 325330 335 Ile Thr Asp Phe Gly Leu Ala Lys Val Gln Gly Asn Gly Ser Phe Met340 345 350 Lys Thr Phe Cys Gly Thr Leu Ala Tyr Val Ala Pro Glu Val IleArg 355 360 365 Gly Lys Asp Thr Ser Val Ser Pro Asp Glu Tyr Glu Glu ArgAsn Glu 370 375 380 Tyr Ser Ser Leu Val Asp Met Trp Ser Met Gly Cys LeuVal Tyr Val 385 390 395 400 Ile Leu Thr Gly His Leu Pro Phe Ser Gly SerThr Gln Asp Gln Leu 405 410 415 Tyr Lys Gln Ile Gly Arg Gly Ser Tyr HisGlu Gly Pro Leu Lys Asp 420 425 430 Phe Arg Ile Ser Glu Glu Ala Arg AspPhe Ile Asp Ser Leu Leu Gln 435 440 445 Val Asp Pro Asn Asn Arg Ser ThrAla Ala Lys Ala Leu Asn His Pro 450 455 460 Trp Ile Lys Met Ser Pro LeuGly Ser Gln Ser Tyr Gly Asp Phe Ser 465 470 475 480 Gln Ile Ser Leu SerGln Ser Leu Ser Gln Gln Lys Leu Leu Glu Asn 485 490 495 Met Asp Asp AlaGln Tyr Glu Phe Val Lys Ala Gln Arg Lys Leu Gln 500 505 510 Met Glu GlnGln Leu Gln Glu Gln Asp Gln Glu Asp Gln Asp Gly Lys 515 520 525 Ile GlnGly Phe Lys Ile Pro Ala His Ala Pro Ile Arg Tyr Thr Gln 530 535 540 ProLys Ser Ile Glu Ala Glu Thr Arg Glu Gln Lys Leu Leu His Ser 545 550 555560 Asn Asn Thr Glu Asn Val Lys Ser Ser Lys Lys Lys Gly Asn Gly Arg 565570 575 Phe Leu Thr Leu Lys Pro Leu Pro Asp Ser Ile Ile Gln Glu Ser Leu580 585 590 Glu Ile Gln Gln Gly Val Asn Pro Phe Phe Ile Gly Arg Ser GluAsp 595 600 605 Cys Asn Cys Lys Ile Glu Asp Asn Arg Leu Ser Arg Val HisCys Phe 610 615 620 Ile Phe Lys Lys Arg His Ala Val Gly Lys Ser Met TyrGlu Ser Pro 625 630 635 640 Ala Gln Gly Leu Asp Asp Ile Trp Tyr Cys HisThr Gly Thr Asn Val 645 650 655 Ser Tyr Leu Asn Asn Asn Arg Met Ile GlnGly Thr Lys Phe Leu Leu 660 665 670 Gln Asp Gly Asp Glu Ile Lys Ile IleTrp Asp Lys Asn Asn Lys Phe 675 680 685 Val Ile Gly Phe Lys Val Glu IleAsn Asp Thr Thr Gly Leu Phe Asn 690 695 700 Glu Gly Leu Gly Met Leu GlnGlu Gln Arg Val Val Leu Lys Gln Thr 705 710 715 720 Ala Glu Glu Lys AspLeu Val Lys Lys Leu Thr Gln Met Met Ala Ala 725 730 735 Gln Arg Ala AsnGln Pro Ser Ala Ser Ser Ser Ser Met Ser Ala Lys 740 745 750 Lys Pro ProVal Ser Asp Thr Asn Asn Asn Gly Asn Asn Ser Val Leu 755 760 765 Asn AspLeu Val Glu Ser Pro Ile Asn Ala Asn Thr Gly Asn Ile Leu 770 775 780 LysArg Ile His Ser Val Ser Leu Ser Gln Ser Gln Ile Asp Pro Ser 785 790 795800 Lys Lys Val Lys Arg Ala Lys Leu Asp Gln Thr Ser Lys Gly Pro Glu 805810 815 Asn Leu Gln Phe Ser 820 <210> SEQ ID NO 17 <211> LENGTH: 2525<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (289)...(1230) <400> SEQUENCE: 17tcctgccccg cggcgctgcc gcacgagccc cacgagccgc tcaccccgcc gttctcagcg 60ctgcccgacc ccgctggcgc gccctcccgc cgccagtccc ggcagcgccc tcagttgtcc 120tccgactcgc cctcggcctt ccgcgccagc cgcagccaca gccgcaacgc cacccgcagc 180cacagccaca gccacagccc caggcatagc cttcggcaca gccccggctc cggctcctgc 240ggcagctcct ctgggcaccg tccctgcgcc gacatcctgg aggttggg atg ctc ttg 297 MetLeu Leu 1 tcc aaa atc aac tcg ctt gcc cac ctg cgc gcc gcg ccc tgc aacgac 345 Ser Lys Ile Asn Ser Leu Ala His Leu Arg Ala Ala Pro Cys Asn Asp5 10 15 ctg cac gcc acc aag ctg gcg ccc ggc aag gag aag gag ccc ctg gag393 Leu His Ala Thr Lys Leu Ala Pro Gly Lys Glu Lys Glu Pro Leu Glu 2025 30 35 tcg cag tac cag gtg ggc ccg cta ctg ggc agc ggc ggc ttc ggc tcg441 Ser Gln Tyr Gln Val Gly Pro Leu Leu Gly Ser Gly Gly Phe Gly Ser 4045 50 gtc tac tca ggc atc cgc gtc tcc gac aac ttg ccg gtg gcc atc aaa489 Val Tyr Ser Gly Ile Arg Val Ser Asp Asn Leu Pro Val Ala Ile Lys 5560 65 cac gtg gag aag gac cgg att tcc gac tgg gga gag ctg cct aat ggc537 His Val Glu Lys Asp Arg Ile Ser Asp Trp Gly Glu Leu Pro Asn Gly 7075 80 act cga gtg ccc atg gaa gtg gtc ctg ctg aag aag gtg agc tcg ggt585 Thr Arg Val Pro Met Glu Val Val Leu Leu Lys Lys Val Ser Ser Gly 8590 95 ttc tcc ggc gtc att agg ctc ctg gac tgg ttc gag agg ccc gac agt633 Phe Ser Gly Val Ile Arg Leu Leu Asp Trp Phe Glu Arg Pro Asp Ser 100105 110 115 ttc gtc ctg atc ctg gag agg ccc gag ccg gtg caa gat ctc ttcgac 681 Phe Val Leu Ile Leu Glu Arg Pro Glu Pro Val Gln Asp Leu Phe Asp120 125 130 ttc atc acg gaa agg gga gcc ctg caa gag gag ctc gcc cgc agcttc 729 Phe Ile Thr Glu Arg Gly Ala Leu Gln Glu Glu Leu Ala Arg Ser Phe135 140 145 ttc tgg cag gtg ctg gag gcc gtg cgg cac tgc cac aac tgc ggggtg 777 Phe Trp Gln Val Leu Glu Ala Val Arg His Cys His Asn Cys Gly Val150 155 160 ctc cac cgc gac atc aag gac gaa aac atc ctt atc gac ctc aatcgc 825 Leu His Arg Asp Ile Lys Asp Glu Asn Ile Leu Ile Asp Leu Asn Arg165 170 175 ggc gag ctc aag ctc atc gac ttc ggg tcg ggg gcg ctg ctc aaagac 873 Gly Glu Leu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu Leu Lys Asp180 185 190 195 acc gtc tac acg gac ttc gat ggg acc cga gtg tat agc cctcca gag 921 Thr Val Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser Pro ProGlu 200 205 210 tgg atc cgc tac cat cgc tac cat ggc agg tcg gcg gca gtctgg tcc 969 Trp Ile Arg Tyr His Arg Tyr His Gly Arg Ser Ala Ala Val TrpSer 215 220 225 ctg ggg atc ctg ctg tat gat atg gtg tgt gga gat att cctttc gag 1017 Leu Gly Ile Leu Leu Tyr Asp Met Val Cys Gly Asp Ile Pro PheGlu 230 235 240 cat gac gaa gag atc atc agg ggc cag gtt ttc ttc agg cagagg gtc 1065 His Asp Glu Glu Ile Ile Arg Gly Gln Val Phe Phe Arg Gln ArgVal 245 250 255 tct tca gaa tgt cag cat ctc att aga tgg tgc ttg gcc ctgaga cca 1113 Ser Ser Glu Cys Gln His Leu Ile Arg Trp Cys Leu Ala Leu ArgPro 260 265 270 275 tca gat agg cca acc ttc gaa gaa atc cag aac cat ccatgg atg caa 1161 Ser Asp Arg Pro Thr Phe Glu Glu Ile Gln Asn His Pro TrpMet Gln 280 285 290 gat gtt ctc ctg ccc cag gaa act gct gag atc cac ctccac agc ctg 1209 Asp Val Leu Leu Pro Gln Glu Thr Ala Glu Ile His Leu HisSer Leu 295 300 305 tcg ccg ggg ccc agc aaa tag cagcctttct ggcaggtcctcccctctctt 1260 Ser Pro Gly Pro Ser Lys * 310 gtcagatgcc cgagggaggggaagcttctg tctccagctt cccgagtacc agtgacacgt 1320 ctcgccaagc aggacagtgcttgatacagg aacaacattt acaactcatt ccagatccca 1380 ggcccctgga ggctgcctcccaacagtgag gaagagtgac tctccagggg tcctaggcct 1440 caactcctcc catagatactctcttcttct cataggtgtc cagcattgct ggactgctga 1500 aatatcccgg gggtggggggtgggggtggg tcagaaccct gccatggaac tgtttccttc 1560 atcatgagtt ctgctgaatgccgcgatggg tcaggtaggg gggaaacagg ttgggatggg 1620 ataggactag caccattttaagtccctgtc acctcttccg actctttctg agtgccttct 1680 gtggggactc cggctgtgctgggagaaata cttgaacttg cctcttttac ctgctgcttc 1740 tccaaaaatc tgcctgggttttgttcccta tttttctctc ctgtcctccc tcaccccctc 1800 cttcatatga aaggtgccatggaagaggct acagggccaa acgctgagcc acctgccctt 1860 ttttctgcct cctttagtaaaactccgagt gaactggtct tcctttttgg tttttactta 1920 actgtttcaa agccaagacctcacacacag aaaaaatgca caaacaatgc aatcaacaga 1980 aaagctgtaa atgtgtgtacagttggcatg gtagtataca aaaagattgt agtggatcta 2040 atttttcaga aattttgcctttaagttatt ttacctgttt ttgtttcttg ttttgaaaga 2100 tgcgcattct aacctggaggtcaatgttat gtatttattt atttatttat ttggttccct 2160 tcctattcca agcttccatagctgctgccc tagttttctt tcctcctttc ctcctctgac 2220 ttggggacct tttgggggagggctgcgacg cttgctctgt ttgtggggtg acgggactca 2280 ggcgggacag tgctgcagctccctggcttc tgtggggccc ctcacctact tacccaggtg 2340 ggtcccggct ctgtgggtgatggggagggg cattgctgac tgtgtatata ggataattat 2400 gaaaagcagt tctggatggtgtgccttcca gatcctctct ggggctgtgt tttgagcagc 2460 aggtagcctg gctggttttatctgagtgaa atactgtaca ggggaataaa agagatctta 2520 ttttt 2525 <210> SEQ IDNO 18 <211> LENGTH: 313 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 18 Met Leu Leu Ser Lys Ile Asn Ser Leu Ala His Leu ArgAla Ala Pro 1 5 10 15 Cys Asn Asp Leu His Ala Thr Lys Leu Ala Pro GlyLys Glu Lys Glu 20 25 30 Pro Leu Glu Ser Gln Tyr Gln Val Gly Pro Leu LeuGly Ser Gly Gly 35 40 45 Phe Gly Ser Val Tyr Ser Gly Ile Arg Val Ser AspAsn Leu Pro Val 50 55 60 Ala Ile Lys His Val Glu Lys Asp Arg Ile Ser AspTrp Gly Glu Leu 65 70 75 80 Pro Asn Gly Thr Arg Val Pro Met Glu Val ValLeu Leu Lys Lys Val 85 90 95 Ser Ser Gly Phe Ser Gly Val Ile Arg Leu LeuAsp Trp Phe Glu Arg 100 105 110 Pro Asp Ser Phe Val Leu Ile Leu Glu ArgPro Glu Pro Val Gln Asp 115 120 125 Leu Phe Asp Phe Ile Thr Glu Arg GlyAla Leu Gln Glu Glu Leu Ala 130 135 140 Arg Ser Phe Phe Trp Gln Val LeuGlu Ala Val Arg His Cys His Asn 145 150 155 160 Cys Gly Val Leu His ArgAsp Ile Lys Asp Glu Asn Ile Leu Ile Asp 165 170 175 Leu Asn Arg Gly GluLeu Lys Leu Ile Asp Phe Gly Ser Gly Ala Leu 180 185 190 Leu Lys Asp ThrVal Tyr Thr Asp Phe Asp Gly Thr Arg Val Tyr Ser 195 200 205 Pro Pro GluTrp Ile Arg Tyr His Arg Tyr His Gly Arg Ser Ala Ala 210 215 220 Val TrpSer Leu Gly Ile Leu Leu Tyr Asp Met Val Cys Gly Asp Ile 225 230 235 240Pro Phe Glu His Asp Glu Glu Ile Ile Arg Gly Gln Val Phe Phe Arg 245 250255 Gln Arg Val Ser Ser Glu Cys Gln His Leu Ile Arg Trp Cys Leu Ala 260265 270 Leu Arg Pro Ser Asp Arg Pro Thr Phe Glu Glu Ile Gln Asn His Pro275 280 285 Trp Met Gln Asp Val Leu Leu Pro Gln Glu Thr Ala Glu Ile HisLeu 290 295 300 His Ser Leu Ser Pro Gly Pro Ser Lys 305 310 <210> SEQ IDNO 19 <211> LENGTH: 3401 <212> TYPE: DNA <213> ORGANISM: Saccharomycescerevisiae <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(789)...(2795) <400> SEQUENCE: 19 atcgatacaa ctccccagaa atatcactgttgagagttca tgtcaagacg tagtatagta 60 ttgcaacagg aaaaaaaaat cttattgcgtagcactctgt atctatttta tatatctgtt 120 tctatatttg attaatctct tgttatcttggtgatgatcg cacaagtatg tactcctgta 180 tctgcaagaa tatctgtttt aaacttttcaaagcaaggaa accccgtctt atataggtta 240 tccgcaaagg tcacattttc ttgcaaatagaagaaaaagc acccacaagc acactaacac 300 agtgccagag caaaactata tcctttgcatccgatctcaa acgctgttct tatcgcatct 360 gtcttcgtcc tttcatctgc atttaccttttctttttcat cctctatttg ccttttcatt 420 agtggcaatt tttccagttt tttccctctgcgtcccgttg cacctgaaag gatctttcta 480 acgtgtgttg tctactagtg agcgatttcgtgagccatac acgttctata gaaaattgaa 540 taaactttac ttcaaaggga tctggacacagagataactg cttacctgct tgccggaaga 600 aaagaattac taaaaaagaa gacaagggtagctgctattg tgggtacacg tttcacagaa 660 ctactttttc cttgtccttc tccagacatcaacgtcatac aactaaaact gataaagtac 720 ccgtttttcc gtacatttct atagatacattattatatta agcagatcga gacgttaatt 780 tctcaaag atg gaa gac aag ttt gctaac ctc agt ctc cat gag aaa act 830 Met Glu Asp Lys Phe Ala Asn Leu SerLeu His Glu Lys Thr 1 5 10 ggt aag tca tct atc caa tta aac gag caa acaggc tca gat aat ggc 878 Gly Lys Ser Ser Ile Gln Leu Asn Glu Gln Thr GlySer Asp Asn Gly 15 20 25 30 tct gct gtc aag aga aca tct tcg acg tcc tcgcac tac aat aac atc 926 Ser Ala Val Lys Arg Thr Ser Ser Thr Ser Ser HisTyr Asn Asn Ile 35 40 45 aac gct gac ctt cat gct cgt gta aaa gct ttt caagaa caa cgt gca 974 Asn Ala Asp Leu His Ala Arg Val Lys Ala Phe Gln GluGln Arg Ala 50 55 60 ttg aaa agg tct gcc agc gtg ggc agt aat caa agc gagcaa gac aaa 1022 Leu Lys Arg Ser Ala Ser Val Gly Ser Asn Gln Ser Glu GlnAsp Lys 65 70 75 ggc agt tca caa tca cct aaa cat att cag cag att gtt aataag cca 1070 Gly Ser Ser Gln Ser Pro Lys His Ile Gln Gln Ile Val Asn LysPro 80 85 90 ttg ccg cct ctt ccc gta gca gga agt tct aag gtt tca caa agaatg 1118 Leu Pro Pro Leu Pro Val Ala Gly Ser Ser Lys Val Ser Gln Arg Met95 100 105 110 agt agc caa gtc gtg caa gcg tcc tcc aag agc act ctt aagaac gtt 1166 Ser Ser Gln Val Val Gln Ala Ser Ser Lys Ser Thr Leu Lys AsnVal 115 120 125 ctg gac aat caa gaa aca caa aac att acc gac gta aat attaac atc 1214 Leu Asp Asn Gln Glu Thr Gln Asn Ile Thr Asp Val Asn Ile AsnIle 130 135 140 gat aca acc aaa att acc gcc aca aca att ggt gta aat actggc cta 1262 Asp Thr Thr Lys Ile Thr Ala Thr Thr Ile Gly Val Asn Thr GlyLeu 145 150 155 cct gct act gac att acg ccg tca gtt tct aat act gca tcagca aca 1310 Pro Ala Thr Asp Ile Thr Pro Ser Val Ser Asn Thr Ala Ser AlaThr 160 165 170 cat aag gcg caa ttg ctg aat cct aac aga agg gca cca agaagg ccg 1358 His Lys Ala Gln Leu Leu Asn Pro Asn Arg Arg Ala Pro Arg ArgPro 175 180 185 190 ctt tct acc cag cac cct aca aga cca aat gtt gcc ccgcat aag gcc 1406 Leu Ser Thr Gln His Pro Thr Arg Pro Asn Val Ala Pro HisLys Ala 195 200 205 cct gct ata atc aac aca cca aaa caa agt tta agt gcccgt cga ggg 1454 Pro Ala Ile Ile Asn Thr Pro Lys Gln Ser Leu Ser Ala ArgArg Gly 210 215 220 ctc aaa tta cca cca gga gga atg tca tta aaa atg cccact aaa aca 1502 Leu Lys Leu Pro Pro Gly Gly Met Ser Leu Lys Met Pro ThrLys Thr 225 230 235 gct caa cag ccg cag cag ttt gcc cca agc cct tca aacaaa aaa cat 1550 Ala Gln Gln Pro Gln Gln Phe Ala Pro Ser Pro Ser Asn LysLys His 240 245 250 ata gaa acc tta tca aac agc aaa gtt gtt gaa ggg aaaaga tcg aat 1598 Ile Glu Thr Leu Ser Asn Ser Lys Val Val Glu Gly Lys ArgSer Asn 255 260 265 270 ccg ggt tct ttg ata aat ggt gtg caa agc aca tccacc tca tca agt 1646 Pro Gly Ser Leu Ile Asn Gly Val Gln Ser Thr Ser ThrSer Ser Ser 275 280 285 acc gaa ggc cca cat gac act gta ggc act aca cccaga act gga aac 1694 Thr Glu Gly Pro His Asp Thr Val Gly Thr Thr Pro ArgThr Gly Asn 290 295 300 agc aac aac tct tca aat tct ggt agt agt ggt ggtggt ggt ctt ttc 1742 Ser Asn Asn Ser Ser Asn Ser Gly Ser Ser Gly Gly GlyGly Leu Phe 305 310 315 gca aat ttc tcg aaa tac gtg gat atc aaa tcc ggctct ttg aat ttt 1790 Ala Asn Phe Ser Lys Tyr Val Asp Ile Lys Ser Gly SerLeu Asn Phe 320 325 330 gca ggc aaa cta tcg cta tcc tct aaa gga ata gatttc agc aat ggt 1838 Ala Gly Lys Leu Ser Leu Ser Ser Lys Gly Ile Asp PheSer Asn Gly 335 340 345 350 tct agt tcg aga att aca ttg gac gaa cta gaattt ttg gat gaa ctg 1886 Ser Ser Ser Arg Ile Thr Leu Asp Glu Leu Glu PheLeu Asp Glu Leu 355 360 365 ggt cat ggt aac tat ggt aac gtc tca aag gtactg cat aag ccc aca 1934 Gly His Gly Asn Tyr Gly Asn Val Ser Lys Val LeuHis Lys Pro Thr 370 375 380 aat gtt att atg gcg acg aag gaa gtc cgt ttggag cta gat gag gct 1982 Asn Val Ile Met Ala Thr Lys Glu Val Arg Leu GluLeu Asp Glu Ala 385 390 395 aaa ttt aga caa att tta atg gaa cta gaa gttttg cat aaa tgc aat 2030 Lys Phe Arg Gln Ile Leu Met Glu Leu Glu Val LeuHis Lys Cys Asn 400 405 410 tct ccc tat att gtg gat ttt tat ggt gca ttcttt att gag ggc gcc 2078 Ser Pro Tyr Ile Val Asp Phe Tyr Gly Ala Phe PheIle Glu Gly Ala 415 420 425 430 gtc tac atg tgt atg gaa tac atg gat ggtggt tcc ttg gat aaa ata 2126 Val Tyr Met Cys Met Glu Tyr Met Asp Gly GlySer Leu Asp Lys Ile 435 440 445 tac gac gaa tca tct gaa atc ggc ggc attgat gaa cct cag cta gcg 2174 Tyr Asp Glu Ser Ser Glu Ile Gly Gly Ile AspGlu Pro Gln Leu Ala 450 455 460 ttt att gcc aat gct gtc att cat gga ctaaaa gaa ctc aaa gag cag 2222 Phe Ile Ala Asn Ala Val Ile His Gly Leu LysGlu Leu Lys Glu Gln 465 470 475 cat aat atc ata cac aga gat gtc aaa ccaaca aat att tta tgt tca 2270 His Asn Ile Ile His Arg Asp Val Lys Pro ThrAsn Ile Leu Cys Ser 480 485 490 gcc aac caa ggc acc gta aag ctg tgc gatttc ggt gtt tct ggt aat 2318 Ala Asn Gln Gly Thr Val Lys Leu Cys Asp PheGly Val Ser Gly Asn 495 500 505 510 ttg gtg gca tct tta gcg aag act aatatt ggt tgt cag tca tac atg 2366 Leu Val Ala Ser Leu Ala Lys Thr Asn IleGly Cys Gln Ser Tyr Met 515 520 525 gca cct gaa cga atc aaa tcg ttg aatcca gat aga gcc acc tat acc 2414 Ala Pro Glu Arg Ile Lys Ser Leu Asn ProAsp Arg Ala Thr Tyr Thr 530 535 540 gta cag tca gac atc tgg tct tta ggttta agc att ctg gaa atg gca 2462 Val Gln Ser Asp Ile Trp Ser Leu Gly LeuSer Ile Leu Glu Met Ala 545 550 555 cta ggt aga tat ccg tat cca cca gaaaca tac gac aac att ttc tct 2510 Leu Gly Arg Tyr Pro Tyr Pro Pro Glu ThrTyr Asp Asn Ile Phe Ser 560 565 570 caa ttg agc gct att gtt gat ggg ccgcca ccg aga tta cct tca gat 2558 Gln Leu Ser Ala Ile Val Asp Gly Pro ProPro Arg Leu Pro Ser Asp 575 580 585 590 aaa ttc agt tct gac gca caa gatttt gtt tct tta tgt cta caa aag 2606 Lys Phe Ser Ser Asp Ala Gln Asp PheVal Ser Leu Cys Leu Gln Lys 595 600 605 att ccg gaa aga aga cct aca tacgca gct tta aca gag cat cct tgg 2654 Ile Pro Glu Arg Arg Pro Thr Tyr AlaAla Leu Thr Glu His Pro Trp 610 615 620 tta gta aaa tac aga aac cag gatgtc cac atg agt gag tat atc act 2702 Leu Val Lys Tyr Arg Asn Gln Asp ValHis Met Ser Glu Tyr Ile Thr 625 630 635 gaa cga tta gaa agg cgc aac aaaatc tta cgg gaa cgt ggt gag aat 2750 Glu Arg Leu Glu Arg Arg Asn Lys IleLeu Arg Glu Arg Gly Glu Asn 640 645 650 ggt tta tct aaa aat gta ccg gcatta cat atg ggt ggt tta tag 2795 Gly Leu Ser Lys Asn Val Pro Ala Leu HisMet Gly Gly Leu * 655 660 665 cgttaatatc caaataaaag caaacaggcacgtgaatata acaacaaaaa aaaagcagac 2855 gaaaagctac tgtggaaatg atgcggcgaatacaaaaaaa ccttacatat acatatgttt 2915 attgtaataa acttgcatta tactcgttatagacatatat atatatatat attcatatat 2975 atatatcgtc tgacttcctt ttgtcgaacctaaaaaaggg cacgaattat gacagagtat 3035 tgaggggatg ttatttcaag caccggcaagtgaagcgatg tggacgtcaa tatattgtgt 3095 tattcgatta ttgctacggc catcgactcctcgaaattat ttacgttcgg ggctgacaac 3155 gcaagaaaga aaaaatgctc tggaattgtctgatggtttt tccgctcttt acggctcaag 3215 gctaggaaag aaaaaaaagt ccaaaatcatcgagaaaata aaaggtgttt tgaaagttca 3275 aatccacgtt attgagagta gatgtggagtctggaccagg aactatacct gtatcttacc 3335 ctaacttcta aattttgcta ctttcacggaaaacagtaaa taattaccta tcaagataaa 3395 gagctc 3401 <210> SEQ ID NO 20<211> LENGTH: 668 <212> TYPE: PRT <213> ORGANISM: Saccharomycescerevisiae <400> SEQUENCE: 20 Met Glu Asp Lys Phe Ala Asn Leu Ser LeuHis Glu Lys Thr Gly Lys 1 5 10 15 Ser Ser Ile Gln Leu Asn Glu Gln ThrGly Ser Asp Asn Gly Ser Ala 20 25 30 Val Lys Arg Thr Ser Ser Thr Ser SerHis Tyr Asn Asn Ile Asn Ala 35 40 45 Asp Leu His Ala Arg Val Lys Ala PheGln Glu Gln Arg Ala Leu Lys 50 55 60 Arg Ser Ala Ser Val Gly Ser Asn GlnSer Glu Gln Asp Lys Gly Ser 65 70 75 80 Ser Gln Ser Pro Lys His Ile GlnGln Ile Val Asn Lys Pro Leu Pro 85 90 95 Pro Leu Pro Val Ala Gly Ser SerLys Val Ser Gln Arg Met Ser Ser 100 105 110 Gln Val Val Gln Ala Ser SerLys Ser Thr Leu Lys Asn Val Leu Asp 115 120 125 Asn Gln Glu Thr Gln AsnIle Thr Asp Val Asn Ile Asn Ile Asp Thr 130 135 140 Thr Lys Ile Thr AlaThr Thr Ile Gly Val Asn Thr Gly Leu Pro Ala 145 150 155 160 Thr Asp IleThr Pro Ser Val Ser Asn Thr Ala Ser Ala Thr His Lys 165 170 175 Ala GlnLeu Leu Asn Pro Asn Arg Arg Ala Pro Arg Arg Pro Leu Ser 180 185 190 ThrGln His Pro Thr Arg Pro Asn Val Ala Pro His Lys Ala Pro Ala 195 200 205Ile Ile Asn Thr Pro Lys Gln Ser Leu Ser Ala Arg Arg Gly Leu Lys 210 215220 Leu Pro Pro Gly Gly Met Ser Leu Lys Met Pro Thr Lys Thr Ala Gln 225230 235 240 Gln Pro Gln Gln Phe Ala Pro Ser Pro Ser Asn Lys Lys His IleGlu 245 250 255 Thr Leu Ser Asn Ser Lys Val Val Glu Gly Lys Arg Ser AsnPro Gly 260 265 270 Ser Leu Ile Asn Gly Val Gln Ser Thr Ser Thr Ser SerSer Thr Glu 275 280 285 Gly Pro His Asp Thr Val Gly Thr Thr Pro Arg ThrGly Asn Ser Asn 290 295 300 Asn Ser Ser Asn Ser Gly Ser Ser Gly Gly GlyGly Leu Phe Ala Asn 305 310 315 320 Phe Ser Lys Tyr Val Asp Ile Lys SerGly Ser Leu Asn Phe Ala Gly 325 330 335 Lys Leu Ser Leu Ser Ser Lys GlyIle Asp Phe Ser Asn Gly Ser Ser 340 345 350 Ser Arg Ile Thr Leu Asp GluLeu Glu Phe Leu Asp Glu Leu Gly His 355 360 365 Gly Asn Tyr Gly Asn ValSer Lys Val Leu His Lys Pro Thr Asn Val 370 375 380 Ile Met Ala Thr LysGlu Val Arg Leu Glu Leu Asp Glu Ala Lys Phe 385 390 395 400 Arg Gln IleLeu Met Glu Leu Glu Val Leu His Lys Cys Asn Ser Pro 405 410 415 Tyr IleVal Asp Phe Tyr Gly Ala Phe Phe Ile Glu Gly Ala Val Tyr 420 425 430 MetCys Met Glu Tyr Met Asp Gly Gly Ser Leu Asp Lys Ile Tyr Asp 435 440 445Glu Ser Ser Glu Ile Gly Gly Ile Asp Glu Pro Gln Leu Ala Phe Ile 450 455460 Ala Asn Ala Val Ile His Gly Leu Lys Glu Leu Lys Glu Gln His Asn 465470 475 480 Ile Ile His Arg Asp Val Lys Pro Thr Asn Ile Leu Cys Ser AlaAsn 485 490 495 Gln Gly Thr Val Lys Leu Cys Asp Phe Gly Val Ser Gly AsnLeu Val 500 505 510 Ala Ser Leu Ala Lys Thr Asn Ile Gly Cys Gln Ser TyrMet Ala Pro 515 520 525 Glu Arg Ile Lys Ser Leu Asn Pro Asp Arg Ala ThrTyr Thr Val Gln 530 535 540 Ser Asp Ile Trp Ser Leu Gly Leu Ser Ile LeuGlu Met Ala Leu Gly 545 550 555 560 Arg Tyr Pro Tyr Pro Pro Glu Thr TyrAsp Asn Ile Phe Ser Gln Leu 565 570 575 Ser Ala Ile Val Asp Gly Pro ProPro Arg Leu Pro Ser Asp Lys Phe 580 585 590 Ser Ser Asp Ala Gln Asp PheVal Ser Leu Cys Leu Gln Lys Ile Pro 595 600 605 Glu Arg Arg Pro Thr TyrAla Ala Leu Thr Glu His Pro Trp Leu Val 610 615 620 Lys Tyr Arg Asn GlnAsp Val His Met Ser Glu Tyr Ile Thr Glu Arg 625 630 635 640 Leu Glu ArgArg Asn Lys Ile Leu Arg Glu Arg Gly Glu Asn Gly Leu 645 650 655 Ser LysAsn Val Pro Ala Leu His Met Gly Gly Leu 660 665 <210> SEQ ID NO 21 <211>LENGTH: 1883 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (121)...(1203) <400> SEQUENCE: 21gtgagccacc gcccccagcc tggcctggca tttctttgag ttcaggaagt gtgacaagga 60tttggacacc cagaaataag cgtgtcgaga agagcacaag cagaggatcc agcgctcggc 120atg gcg gag cca gat ctg gag tgc gag cag atc cgt ctg aag tgt att 168 MetAla Glu Pro Asp Leu Glu Cys Glu Gln Ile Arg Leu Lys Cys Ile 1 5 10 15cgt aag gag ggc ttc ttc acg gtg cct ccg gaa cac agg ctg gga cga 216 ArgLys Glu Gly Phe Phe Thr Val Pro Pro Glu His Arg Leu Gly Arg 20 25 30 tgccgg agt gtg aag gag ttt gag aag ctg aac cgc att gga gag ggt 264 Cys ArgSer Val Lys Glu Phe Glu Lys Leu Asn Arg Ile Gly Glu Gly 35 40 45 acc tacggc att gtg tat cgg gcc cgg gac acc cag aca gat gag att 312 Thr Tyr GlyIle Val Tyr Arg Ala Arg Asp Thr Gln Thr Asp Glu Ile 50 55 60 gtc gca ctgaag aag gtg cgg atg gac aag gag aag gat ggc atc ccc 360 Val Ala Leu LysLys Val Arg Met Asp Lys Glu Lys Asp Gly Ile Pro 65 70 75 80 atc agc agcttg cgg gag atc acg ctg ctg ctc cgc ctg cgt cat ccg 408 Ile Ser Ser LeuArg Glu Ile Thr Leu Leu Leu Arg Leu Arg His Pro 85 90 95 aac atc gtg gagctg aag gag gtg gtt gtg ggg aac cac ctg gag agc 456 Asn Ile Val Glu LeuLys Glu Val Val Val Gly Asn His Leu Glu Ser 100 105 110 atc ttc ctg gtgatg ggt tac tgt gag cag gac ctg gcc agc ctc ctg 504 Ile Phe Leu Val MetGly Tyr Cys Glu Gln Asp Leu Ala Ser Leu Leu 115 120 125 gag aat atg ccaaca ccc ttc tcg gag gct cag gtc aag tgc atc gtg 552 Glu Asn Met Pro ThrPro Phe Ser Glu Ala Gln Val Lys Cys Ile Val 130 135 140 ctg cag gtg ctccgg ggc ctc cag tat ctg cac agg aac ttc att atc 600 Leu Gln Val Leu ArgGly Leu Gln Tyr Leu His Arg Asn Phe Ile Ile 145 150 155 160 cac agg gacctg aag gtt tcc aac ttg ctc atg acc gac aag ggt tgt 648 His Arg Asp LeuLys Val Ser Asn Leu Leu Met Thr Asp Lys Gly Cys 165 170 175 gtg aag acagcg gat ttc ggc ctg gcc cgg gcc tat ggt gtc cca gta 696 Val Lys Thr AlaAsp Phe Gly Leu Ala Arg Ala Tyr Gly Val Pro Val 180 185 190 aag cca atgacc ccc aag gtg gtc act ctc tgg tac cga gcc cct gaa 744 Lys Pro Met ThrPro Lys Val Val Thr Leu Trp Tyr Arg Ala Pro Glu 195 200 205 ctg ctg ttggga acc acc acg cag acc acc agc atc gac atg tgg gct 792 Leu Leu Leu GlyThr Thr Thr Gln Thr Thr Ser Ile Asp Met Trp Ala 210 215 220 gtg ggc tgcata ctg gcc gag ctg ctg gcg cac agg cct ctt ctc ccc 840 Val Gly Cys IleLeu Ala Glu Leu Leu Ala His Arg Pro Leu Leu Pro 225 230 235 240 ggc acttcc gag atc cac cag atc gac ttg atc gtg cag ctg ctg ggc 888 Gly Thr SerGlu Ile His Gln Ile Asp Leu Ile Val Gln Leu Leu Gly 245 250 255 acg cccagt gag aac atc tgg ccg ggc ttt tcc aag ctg cca ctg gtc 936 Thr Pro SerGlu Asn Ile Trp Pro Gly Phe Ser Lys Leu Pro Leu Val 260 265 270 ggc cagtac agc ctc cgg aag cag ccc tac aac aac ctg aag cac aag 984 Gly Gln TyrSer Leu Arg Lys Gln Pro Tyr Asn Asn Leu Lys His Lys 275 280 285 ttc ccatgg ctg tcg gag gcc ggg ctg cgc ctg ctg cac ttc ctg ttc 1032 Phe Pro TrpLeu Ser Glu Ala Gly Leu Arg Leu Leu His Phe Leu Phe 290 295 300 atg tacgac cct aag aaa agg gcg acg gcc ggg gac tgc ctg gag agc 1080 Met Tyr AspPro Lys Lys Arg Ala Thr Ala Gly Asp Cys Leu Glu Ser 305 310 315 320 tcctat ttc aag gag aag ccc cta ccc tgt gag ccg gag ctc atg ccg 1128 Ser TyrPhe Lys Glu Lys Pro Leu Pro Cys Glu Pro Glu Leu Met Pro 325 330 335 accttt ccc cac cac cgc aac aag cgg gcc gcc cca gcc acc tcc gag 1176 Thr PhePro His His Arg Asn Lys Arg Ala Ala Pro Ala Thr Ser Glu 340 345 350 ggccag agc aag cgc tgt aaa ccc tga cggtgggcct ggcacacgcc 1223 Gly Gln SerLys Arg Cys Lys Pro * 355 360 tgtattccca caccaggtct tccgatcagtggtgtctgtg aagggtgccg cgagccaggc 1283 tgaccaggcg cccgggatcc agctcatccccttggctggg aacatcctcc actgacttcc 1343 tcccactgtc tgccctgaac ccactgctgcccccagaaaa aggccgggtg acaccggggg 1403 ctcccagccc gtgcaccctg gaagggcaggtctggcggct ccatccgtgg ctgcaggggt 1463 ctcatgtggt cctcctcgct atgttggaaatgtgcaacca ctgcttcttg ggaggagtgg 1523 tgggtgcagt ccccccgctg tctttgagttgtggtggacc gctggcctgg gatgagaggg 1583 cccagaagac cttcgtatcc cctctcagtcgcccggggct gtcccgtgca tgggttggct 1643 gtggggaccc caggtgggcc tggcaggactccagatgagg acaagaggga caaggtatgg 1703 ggtgggagcc acaattgagg ataccccgagctaccaggag agccctgggc tggaggctga 1763 gctggatccc tgctccccac acggaggacccaacaggagg ccgtggctct gatgctgagc 1823 gaagctatag gctcttgttg gataaaagcttttttaacag aaaaaaaaaa aaaaaaaaaa 1883 <210> SEQ ID NO 22 <211> LENGTH:360 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 MetAla Glu Pro Asp Leu Glu Cys Glu Gln Ile Arg Leu Lys Cys Ile 1 5 10 15Arg Lys Glu Gly Phe Phe Thr Val Pro Pro Glu His Arg Leu Gly Arg 20 25 30Cys Arg Ser Val Lys Glu Phe Glu Lys Leu Asn Arg Ile Gly Glu Gly 35 40 45Thr Tyr Gly Ile Val Tyr Arg Ala Arg Asp Thr Gln Thr Asp Glu Ile 50 55 60Val Ala Leu Lys Lys Val Arg Met Asp Lys Glu Lys Asp Gly Ile Pro 65 70 7580 Ile Ser Ser Leu Arg Glu Ile Thr Leu Leu Leu Arg Leu Arg His Pro 85 9095 Asn Ile Val Glu Leu Lys Glu Val Val Val Gly Asn His Leu Glu Ser 100105 110 Ile Phe Leu Val Met Gly Tyr Cys Glu Gln Asp Leu Ala Ser Leu Leu115 120 125 Glu Asn Met Pro Thr Pro Phe Ser Glu Ala Gln Val Lys Cys IleVal 130 135 140 Leu Gln Val Leu Arg Gly Leu Gln Tyr Leu His Arg Asn PheIle Ile 145 150 155 160 His Arg Asp Leu Lys Val Ser Asn Leu Leu Met ThrAsp Lys Gly Cys 165 170 175 Val Lys Thr Ala Asp Phe Gly Leu Ala Arg AlaTyr Gly Val Pro Val 180 185 190 Lys Pro Met Thr Pro Lys Val Val Thr LeuTrp Tyr Arg Ala Pro Glu 195 200 205 Leu Leu Leu Gly Thr Thr Thr Gln ThrThr Ser Ile Asp Met Trp Ala 210 215 220 Val Gly Cys Ile Leu Ala Glu LeuLeu Ala His Arg Pro Leu Leu Pro 225 230 235 240 Gly Thr Ser Glu Ile HisGln Ile Asp Leu Ile Val Gln Leu Leu Gly 245 250 255 Thr Pro Ser Glu AsnIle Trp Pro Gly Phe Ser Lys Leu Pro Leu Val 260 265 270 Gly Gln Tyr SerLeu Arg Lys Gln Pro Tyr Asn Asn Leu Lys His Lys 275 280 285 Phe Pro TrpLeu Ser Glu Ala Gly Leu Arg Leu Leu His Phe Leu Phe 290 295 300 Met TyrAsp Pro Lys Lys Arg Ala Thr Ala Gly Asp Cys Leu Glu Ser 305 310 315 320Ser Tyr Phe Lys Glu Lys Pro Leu Pro Cys Glu Pro Glu Leu Met Pro 325 330335 Thr Phe Pro His His Arg Asn Lys Arg Ala Ala Pro Ala Thr Ser Glu 340345 350 Gly Gln Ser Lys Arg Cys Lys Pro 355 360 <210> SEQ ID NO 23 <211>LENGTH: 2299 <212> TYPE: DNA <213> ORGANISM: Mus musculus <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (142)...(1698) <400> SEQUENCE: 23ccggacttcc atgggcagca gcagcggcag ggaacggagg gcgaatagat ttcagagcct 60gcacctgaag tacaattcga atcctgctcc agggagcgag ccactgtccg gatccagaaa 120ctttggccac tgggaggaaa a atg gcc agt gat acc cca ggt ttc tac atg 171 MetAla Ser Asp Thr Pro Gly Phe Tyr Met 1 5 10 gac aaa ctt aat aaa tac cgccag atg cac gga gta gcc att acg tat 219 Asp Lys Leu Asn Lys Tyr Arg GlnMet His Gly Val Ala Ile Thr Tyr 15 20 25 aaa gaa ctt agt act tcg gga cctcca cat gac aga agg ttt aca ttt 267 Lys Glu Leu Ser Thr Ser Gly Pro ProHis Asp Arg Arg Phe Thr Phe 30 35 40 caa gtt tta ata gat gag aag gaa tttgga gaa gcc aaa ggt aga tca 315 Gln Val Leu Ile Asp Glu Lys Glu Phe GlyGlu Ala Lys Gly Arg Ser 45 50 55 aag acg gag gca aga aac gct gca gcc aaatta gct gtt gat ata ctt 363 Lys Thr Glu Ala Arg Asn Ala Ala Ala Lys LeuAla Val Asp Ile Leu 60 65 70 gat aac gaa aac aag gtg gat tgt cac acg agtgca tgt gag caa ggc 411 Asp Asn Glu Asn Lys Val Asp Cys His Thr Ser AlaCys Glu Gln Gly 75 80 85 90 ttg ttc gtt ggt aac tac ata ggc ctt gtc aatagc ttt gcc cag aag 459 Leu Phe Val Gly Asn Tyr Ile Gly Leu Val Asn SerPhe Ala Gln Lys 95 100 105 aaa aag ctg tct gta aat tat gaa cag tgt gagccc aac tct gag ttg 507 Lys Lys Leu Ser Val Asn Tyr Glu Gln Cys Glu ProAsn Ser Glu Leu 110 115 120 cct caa aga ttt att tgt aaa tgc aaa att gggcag aca atg tat ggt 555 Pro Gln Arg Phe Ile Cys Lys Cys Lys Ile Gly GlnThr Met Tyr Gly 125 130 135 act ggt tca ggt gtc acc aaa cag gag gca aagcag ttg gct gcg aaa 603 Thr Gly Ser Gly Val Thr Lys Gln Glu Ala Lys GlnLeu Ala Ala Lys 140 145 150 gaa gcc tat cag aag ctg tta aag agc ccg ccgaaa act gcc gga aca 651 Glu Ala Tyr Gln Lys Leu Leu Lys Ser Pro Pro LysThr Ala Gly Thr 155 160 165 170 tcc tct agc gtt gtc aca tct aca ttc agtggc ttt tcc agc agc tcg 699 Ser Ser Ser Val Val Thr Ser Thr Phe Ser GlyPhe Ser Ser Ser Ser 175 180 185 tct atg aca agt aat ggt gtt tcc cag tcagca cct gga agt ttt tcc 747 Ser Met Thr Ser Asn Gly Val Ser Gln Ser AlaPro Gly Ser Phe Ser 190 195 200 tca gag aac gtg ttt acg aac ggt ctc ggagaa aat aaa agg aaa tca 795 Ser Glu Asn Val Phe Thr Asn Gly Leu Gly GluAsn Lys Arg Lys Ser 205 210 215 gga gta aaa gta tcc cct gat gat gtg caaaga aat aaa tat acc ttg 843 Gly Val Lys Val Ser Pro Asp Asp Val Gln ArgAsn Lys Tyr Thr Leu 220 225 230 gac gcc agg ttt aac agc gat ttt gaa gacata gaa gaa att ggc tta 891 Asp Ala Arg Phe Asn Ser Asp Phe Glu Asp IleGlu Glu Ile Gly Leu 235 240 245 250 ggt gga ttt ggt caa gtt ttc aaa gcgaaa cac aga att gat gga aag 939 Gly Gly Phe Gly Gln Val Phe Lys Ala LysHis Arg Ile Asp Gly Lys 255 260 265 aga tac gct att aag cgc gtt aaa tataac acg gag aag gcg gag cac 987 Arg Tyr Ala Ile Lys Arg Val Lys Tyr AsnThr Glu Lys Ala Glu His 270 275 280 gaa gta caa gcg ctg gca gaa ctc aatcac gtc aac att gtc caa tac 1035 Glu Val Gln Ala Leu Ala Glu Leu Asn HisVal Asn Ile Val Gln Tyr 285 290 295 cat agt tgt tgg gag gga gtt gac tatgat cct gag cac agc atg agt 1083 His Ser Cys Trp Glu Gly Val Asp Tyr AspPro Glu His Ser Met Ser 300 305 310 gat aca agt cga tac aaa acc cgg tgcctc ttt att caa atg gaa ttc 1131 Asp Thr Ser Arg Tyr Lys Thr Arg Cys LeuPhe Ile Gln Met Glu Phe 315 320 325 330 tgt gat aaa gga act ttg gag caatgg atg aga aac aga aat cag agt 1179 Cys Asp Lys Gly Thr Leu Glu Gln TrpMet Arg Asn Arg Asn Gln Ser 335 340 345 aaa gtg gac aaa gct ttg att ttggac tta tat gaa caa atc gtg acc 1227 Lys Val Asp Lys Ala Leu Ile Leu AspLeu Tyr Glu Gln Ile Val Thr 350 355 360 gga gtg gag tat ata cac tcg aaaggg tta att cac aga gat ctt aag 1275 Gly Val Glu Tyr Ile His Ser Lys GlyLeu Ile His Arg Asp Leu Lys 365 370 375 cca ggt aat ata ttt tta gta gatgaa aga cac att aag atc gga gac 1323 Pro Gly Asn Ile Phe Leu Val Asp GluArg His Ile Lys Ile Gly Asp 380 385 390 ttt ggc ctt gca aca gcc ctg gaaaat gat gga aaa tcc cga aca agg 1371 Phe Gly Leu Ala Thr Ala Leu Glu AsnAsp Gly Lys Ser Arg Thr Arg 395 400 405 410 aga aca gga act ctt caa tacatg agt cca gaa cag tta ttt tta aag 1419 Arg Thr Gly Thr Leu Gln Tyr MetSer Pro Glu Gln Leu Phe Leu Lys 415 420 425 cac tat gga aaa gaa gtg gacatc ttt gct ttg ggc ctt att cta gct 1467 His Tyr Gly Lys Glu Val Asp IlePhe Ala Leu Gly Leu Ile Leu Ala 430 435 440 gaa ctt ctt cac acg tgc ttcacg gag tca gag aaa ata aag ttt ttc 1515 Glu Leu Leu His Thr Cys Phe ThrGlu Ser Glu Lys Ile Lys Phe Phe 445 450 455 gaa agt cta aga aaa ggc gacttc tct aat gat ata ttc gac aac aaa 1563 Glu Ser Leu Arg Lys Gly Asp PheSer Asn Asp Ile Phe Asp Asn Lys 460 465 470 gaa aaa agc ctt cta aaa aaacta ctc tca gag aaa ccc aag gac cga 1611 Glu Lys Ser Leu Leu Lys Lys LeuLeu Ser Glu Lys Pro Lys Asp Arg 475 480 485 490 cct gag aca tct gaa atcctg aag acc ttg gct gaa tgg agg aac atc 1659 Pro Glu Thr Ser Glu Ile LeuLys Thr Leu Ala Glu Trp Arg Asn Ile 495 500 505 tca gag aaa gaa aag aaacac atg tta ggg cct ttc tga gaaaacattc 1708 Ser Glu Lys Glu Lys Lys HisMet Leu Gly Pro Phe * 510 515 cttctgccgt ggttttcctt taacgatctgcagtctgagg ggagtatcag tgaatattat 1768 ccttcttttc ttaataccac tctcccagacaggttttggt tagggtgacc cacagacatt 1828 gtatttatta ggctatgaaa aagtatgcccatttcctcaa ttgttaattg ctgggcctgt 1888 ggctggctag ctagccaaat atgtaaatgcttgtttctcg tctgcccaaa gagaaaggca 1948 ggctcctgtg tgggaagtca cagagcccccaaagccaact ggatgaggaa ggactctggc 2008 ttttggcata aaaaagagct ggtagtcagagctggggcag aaggtcctgc agacagacag 2068 acagacagac agacagagac acaaagacatggactagaat ggaggaggga gggaggaagg 2128 gagggaggga gagagagaga gagaaagaaagagagagaga ggacatggag acaaaatggc 2188 ttaagttagc tgggctacct gagagactgtcccagaaaac aggccaacaa ccttccttat 2248 gctatataga tgtctcagtg tctttatcattaaacaccaa gcaggagtgc t 2299 <210> SEQ ID NO 24 <211> LENGTH: 518 <212>TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 24 Met Ala SerAsp Thr Pro Gly Phe Tyr Met Asp Lys Leu Asn Lys Tyr 1 5 10 15 Arg GlnMet His Gly Val Ala Ile Thr Tyr Lys Glu Leu Ser Thr Ser 20 25 30 Gly ProPro His Asp Arg Arg Phe Thr Phe Gln Val Leu Ile Asp Glu 35 40 45 Lys GluPhe Gly Glu Ala Lys Gly Arg Ser Lys Thr Glu Ala Arg Asn 50 55 60 Ala AlaAla Lys Leu Ala Val Asp Ile Leu Asp Asn Glu Asn Lys Val 65 70 75 80 AspCys His Thr Ser Ala Cys Glu Gln Gly Leu Phe Val Gly Asn Tyr 85 90 95 IleGly Leu Val Asn Ser Phe Ala Gln Lys Lys Lys Leu Ser Val Asn 100 105 110Tyr Glu Gln Cys Glu Pro Asn Ser Glu Leu Pro Gln Arg Phe Ile Cys 115 120125 Lys Cys Lys Ile Gly Gln Thr Met Tyr Gly Thr Gly Ser Gly Val Thr 130135 140 Lys Gln Glu Ala Lys Gln Leu Ala Ala Lys Glu Ala Tyr Gln Lys Leu145 150 155 160 Leu Lys Ser Pro Pro Lys Thr Ala Gly Thr Ser Ser Ser ValVal Thr 165 170 175 Ser Thr Phe Ser Gly Phe Ser Ser Ser Ser Ser Met ThrSer Asn Gly 180 185 190 Val Ser Gln Ser Ala Pro Gly Ser Phe Ser Ser GluAsn Val Phe Thr 195 200 205 Asn Gly Leu Gly Glu Asn Lys Arg Lys Ser GlyVal Lys Val Ser Pro 210 215 220 Asp Asp Val Gln Arg Asn Lys Tyr Thr LeuAsp Ala Arg Phe Asn Ser 225 230 235 240 Asp Phe Glu Asp Ile Glu Glu IleGly Leu Gly Gly Phe Gly Gln Val 245 250 255 Phe Lys Ala Lys His Arg IleAsp Gly Lys Arg Tyr Ala Ile Lys Arg 260 265 270 Val Lys Tyr Asn Thr GluLys Ala Glu His Glu Val Gln Ala Leu Ala 275 280 285 Glu Leu Asn His ValAsn Ile Val Gln Tyr His Ser Cys Trp Glu Gly 290 295 300 Val Asp Tyr AspPro Glu His Ser Met Ser Asp Thr Ser Arg Tyr Lys 305 310 315 320 Thr ArgCys Leu Phe Ile Gln Met Glu Phe Cys Asp Lys Gly Thr Leu 325 330 335 GluGln Trp Met Arg Asn Arg Asn Gln Ser Lys Val Asp Lys Ala Leu 340 345 350Ile Leu Asp Leu Tyr Glu Gln Ile Val Thr Gly Val Glu Tyr Ile His 355 360365 Ser Lys Gly Leu Ile His Arg Asp Leu Lys Pro Gly Asn Ile Phe Leu 370375 380 Val Asp Glu Arg His Ile Lys Ile Gly Asp Phe Gly Leu Ala Thr Ala385 390 395 400 Leu Glu Asn Asp Gly Lys Ser Arg Thr Arg Arg Thr Gly ThrLeu Gln 405 410 415 Tyr Met Ser Pro Glu Gln Leu Phe Leu Lys His Tyr GlyLys Glu Val 420 425 430 Asp Ile Phe Ala Leu Gly Leu Ile Leu Ala Glu LeuLeu His Thr Cys 435 440 445 Phe Thr Glu Ser Glu Lys Ile Lys Phe Phe GluSer Leu Arg Lys Gly 450 455 460 Asp Phe Ser Asn Asp Ile Phe Asp Asn LysGlu Lys Ser Leu Leu Lys 465 470 475 480 Lys Leu Leu Ser Glu Lys Pro LysAsp Arg Pro Glu Thr Ser Glu Ile 485 490 495 Leu Lys Thr Leu Ala Glu TrpArg Asn Ile Ser Glu Lys Glu Lys Lys 500 505 510 His Met Leu Gly Pro Phe515 <210> SEQ ID NO 25 <211> LENGTH: 2055 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (211)...(1632) <400> SEQUENCE: 25 caggaagact ctgagtccgacgttggccta cccagtcgga aggcagagct gcaatctagt 60 taactacctc ctttcccctagatttccttt cattctgctc aagtcttcgc ctgtgtccga 120 tccctatcta ctttctctcctcttgtagca agcctcagac tccaggcttg agctaggttt 180 tgtttttctc ctggtgagaattcgaagacc atg tct acg gaa ctc ttc tca tcc 234 Met Ser Thr Glu Leu PheSer Ser 1 5 aca aga gag gaa gga agc tct ggc tca gga ccc agt ttt agg tctaat 282 Thr Arg Glu Glu Gly Ser Ser Gly Ser Gly Pro Ser Phe Arg Ser Asn10 15 20 caa agg aaa atg tta aac ctg ctc ctg gag aga gac act tcc ttt acc330 Gln Arg Lys Met Leu Asn Leu Leu Leu Glu Arg Asp Thr Ser Phe Thr 2530 35 40 gtc tgt cca gat gtc cct aga act cca gtg ggc aaa ttt ctt ggt gat378 Val Cys Pro Asp Val Pro Arg Thr Pro Val Gly Lys Phe Leu Gly Asp 4550 55 tct gca aac cta agc att ttg tct gga gga acc cca aaa tgt tgc ctc426 Ser Ala Asn Leu Ser Ile Leu Ser Gly Gly Thr Pro Lys Cys Cys Leu 6065 70 gat ctt tcg aat ctt agc agt ggg gag ata act gcc act cag ctt acc474 Asp Leu Ser Asn Leu Ser Ser Gly Glu Ile Thr Ala Thr Gln Leu Thr 7580 85 act tct gca gac ctt gat gaa act ggt cac ctg gat tct tca gga ctt522 Thr Ser Ala Asp Leu Asp Glu Thr Gly His Leu Asp Ser Ser Gly Leu 9095 100 cag gaa gtg cat tta gct ggg atg aat cat gac cag cac cta atg aaa570 Gln Glu Val His Leu Ala Gly Met Asn His Asp Gln His Leu Met Lys 105110 115 120 tgt agc cca gca cag ctt ctt tgt agc act ccg aat ggt ttg gaccgt 618 Cys Ser Pro Ala Gln Leu Leu Cys Ser Thr Pro Asn Gly Leu Asp Arg125 130 135 ggc cat aga aag aga gat gca atg tgt agt tca tct gca aat aaagaa 666 Gly His Arg Lys Arg Asp Ala Met Cys Ser Ser Ser Ala Asn Lys Glu140 145 150 aat gac aat gga aac ttg gtg gac agt gaa atg aaa tat ttg ggcagt 714 Asn Asp Asn Gly Asn Leu Val Asp Ser Glu Met Lys Tyr Leu Gly Ser155 160 165 ccc att act act gtt cca aaa ttg gat aaa aat cca aac cta ggagaa 762 Pro Ile Thr Thr Val Pro Lys Leu Asp Lys Asn Pro Asn Leu Gly Glu170 175 180 gac cag gca gaa gag att tca gat gaa tta atg gag ttt tcc ctgaaa 810 Asp Gln Ala Glu Glu Ile Ser Asp Glu Leu Met Glu Phe Ser Leu Lys185 190 195 200 gat caa gaa gca aag gtg agc aga agt ggc cta tat cgc tccccg tcg 858 Asp Gln Glu Ala Lys Val Ser Arg Ser Gly Leu Tyr Arg Ser ProSer 205 210 215 atg cca gag aac ttg aac agg cca aga ctg aag cag gtg gaaaaa ttc 906 Met Pro Glu Asn Leu Asn Arg Pro Arg Leu Lys Gln Val Glu LysPhe 220 225 230 aag gac aac aca ata cca gat aaa gtt aaa aaa aag tat ttttct ggc 954 Lys Asp Asn Thr Ile Pro Asp Lys Val Lys Lys Lys Tyr Phe SerGly 235 240 245 caa gga aag ctc agg aag ggc tta tgt tta aag aag aca gtctct ctg 1002 Gln Gly Lys Leu Arg Lys Gly Leu Cys Leu Lys Lys Thr Val SerLeu 250 255 260 tgt gac att act atc act cag atg ctg gag gaa gat tct aaccag ggg 1050 Cys Asp Ile Thr Ile Thr Gln Met Leu Glu Glu Asp Ser Asn GlnGly 265 270 275 280 cac ctg att ggt gat ttt tcc aag gta tgt gcg ctg ccaacc gtg tca 1098 His Leu Ile Gly Asp Phe Ser Lys Val Cys Ala Leu Pro ThrVal Ser 285 290 295 ggg aaa cac caa gat ctg aag tat gtc aac cca gaa acagtg gct gcc 1146 Gly Lys His Gln Asp Leu Lys Tyr Val Asn Pro Glu Thr ValAla Ala 300 305 310 tta ctg tcg ggg aag ttc cag ggt ctg att gag aag ttttat gtc att 1194 Leu Leu Ser Gly Lys Phe Gln Gly Leu Ile Glu Lys Phe TyrVal Ile 315 320 325 gat tgt cgc tat cca tat gag tat ctg gga gga cac atccag gga gcc 1242 Asp Cys Arg Tyr Pro Tyr Glu Tyr Leu Gly Gly His Ile GlnGly Ala 330 335 340 tta aac tta tat agt cag gaa gaa ctg ttt aac ttc tttctg aag aag 1290 Leu Asn Leu Tyr Ser Gln Glu Glu Leu Phe Asn Phe Phe LeuLys Lys 345 350 355 360 ccc atc gtc cct ttg gac acc cag aag aga ata atcatc gtg ttc cac 1338 Pro Ile Val Pro Leu Asp Thr Gln Lys Arg Ile Ile IleVal Phe His 365 370 375 tgt gaa ttc tcc tca gag agg ggc ccc cga atg tgccgc tgt ctg cgt 1386 Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met Cys ArgCys Leu Arg 380 385 390 gaa gag gac agg tct ctg aac cag tat cct gca ttgtac tac cca gag 1434 Glu Glu Asp Arg Ser Leu Asn Gln Tyr Pro Ala Leu TyrTyr Pro Glu 395 400 405 cta tat atc ctt aaa ggc ggc tac aga gac ttc tttcca gaa tat atg 1482 Leu Tyr Ile Leu Lys Gly Gly Tyr Arg Asp Phe Phe ProGlu Tyr Met 410 415 420 gaa ctg tgt gaa cca cag agc tac tgc cct atg catcat cag gac cac 1530 Glu Leu Cys Glu Pro Gln Ser Tyr Cys Pro Met His HisGln Asp His 425 430 435 440 aag act gag ttg ctg agg tgt cga agc cag agcaaa gtg cag gaa ggg 1578 Lys Thr Glu Leu Leu Arg Cys Arg Ser Gln Ser LysVal Gln Glu Gly 445 450 455 gag cgg cag ctg cgg gag cag att gcc ctt ctggtg aag gac atg agc 1626 Glu Arg Gln Leu Arg Glu Gln Ile Ala Leu Leu ValLys Asp Met Ser 460 465 470 cca tga taacattcca gccactggct gctaacaagtcaccaaaaag acactgcaga 1682 Pro * aaccctgagc agaaagaggc cttctggatggccaaaccca agattattaa aagatgtctc 1742 tgcaaaccaa caggctacca acttgtatccaggcctggga atggattagg tttcagcaga 1802 gctgaaagct ggtggcagag tcctggagctggctctataa ggcagccttg agttgcatag 1862 agatttgtat tggttcaggg aactctggcattccttttcc caactcctca tgtcttctca 1922 caagccagcc aactctttct ctctgggcttcgggctatgc aagagcgttg tctaccttct 1982 ttctttgtat tttccttctt tgtttccccctctttctttt ttaaaaatgg aaaaataaac 2042 actacagaat gag 2055 <210> SEQ IDNO 26 <211> LENGTH: 473 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 26 Met Ser Thr Glu Leu Phe Ser Ser Thr Arg Glu Glu GlySer Ser Gly 1 5 10 15 Ser Gly Pro Ser Phe Arg Ser Asn Gln Arg Lys MetLeu Asn Leu Leu 20 25 30 Leu Glu Arg Asp Thr Ser Phe Thr Val Cys Pro AspVal Pro Arg Thr 35 40 45 Pro Val Gly Lys Phe Leu Gly Asp Ser Ala Asn LeuSer Ile Leu Ser 50 55 60 Gly Gly Thr Pro Lys Cys Cys Leu Asp Leu Ser AsnLeu Ser Ser Gly 65 70 75 80 Glu Ile Thr Ala Thr Gln Leu Thr Thr Ser AlaAsp Leu Asp Glu Thr 85 90 95 Gly His Leu Asp Ser Ser Gly Leu Gln Glu ValHis Leu Ala Gly Met 100 105 110 Asn His Asp Gln His Leu Met Lys Cys SerPro Ala Gln Leu Leu Cys 115 120 125 Ser Thr Pro Asn Gly Leu Asp Arg GlyHis Arg Lys Arg Asp Ala Met 130 135 140 Cys Ser Ser Ser Ala Asn Lys GluAsn Asp Asn Gly Asn Leu Val Asp 145 150 155 160 Ser Glu Met Lys Tyr LeuGly Ser Pro Ile Thr Thr Val Pro Lys Leu 165 170 175 Asp Lys Asn Pro AsnLeu Gly Glu Asp Gln Ala Glu Glu Ile Ser Asp 180 185 190 Glu Leu Met GluPhe Ser Leu Lys Asp Gln Glu Ala Lys Val Ser Arg 195 200 205 Ser Gly LeuTyr Arg Ser Pro Ser Met Pro Glu Asn Leu Asn Arg Pro 210 215 220 Arg LeuLys Gln Val Glu Lys Phe Lys Asp Asn Thr Ile Pro Asp Lys 225 230 235 240Val Lys Lys Lys Tyr Phe Ser Gly Gln Gly Lys Leu Arg Lys Gly Leu 245 250255 Cys Leu Lys Lys Thr Val Ser Leu Cys Asp Ile Thr Ile Thr Gln Met 260265 270 Leu Glu Glu Asp Ser Asn Gln Gly His Leu Ile Gly Asp Phe Ser Lys275 280 285 Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gln Asp Leu LysTyr 290 295 300 Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys PheGln Gly 305 310 315 320 Leu Ile Glu Lys Phe Tyr Val Ile Asp Cys Arg TyrPro Tyr Glu Tyr 325 330 335 Leu Gly Gly His Ile Gln Gly Ala Leu Asn LeuTyr Ser Gln Glu Glu 340 345 350 Leu Phe Asn Phe Phe Leu Lys Lys Pro IleVal Pro Leu Asp Thr Gln 355 360 365 Lys Arg Ile Ile Ile Val Phe His CysGlu Phe Ser Ser Glu Arg Gly 370 375 380 Pro Arg Met Cys Arg Cys Leu ArgGlu Glu Asp Arg Ser Leu Asn Gln 385 390 395 400 Tyr Pro Ala Leu Tyr TyrPro Glu Leu Tyr Ile Leu Lys Gly Gly Tyr 405 410 415 Arg Asp Phe Phe ProGlu Tyr Met Glu Leu Cys Glu Pro Gln Ser Tyr 420 425 430 Cys Pro Met HisHis Gln Asp His Lys Thr Glu Leu Leu Arg Cys Arg 435 440 445 Ser Gln SerLys Val Gln Glu Gly Glu Arg Gln Leu Arg Glu Gln Ile 450 455 460 Ala LeuLeu Val Lys Asp Met Ser Pro 465 470 <210> SEQ ID NO 27 <211> LENGTH: 17<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 27 Ser GlyLeu Tyr Arg Ser Pro Ser Met Pro Glu Asn Leu Asn Arg Pro 1 5 10 15 Arg<210> SEQ ID NO 28 <211> LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Homosapiens <400> SEQUENCE: 28 Gly Leu Tyr Arg Ser Pro Ser Met Pro Glu AsnLeu Asn Arg 1 5 10 <210> SEQ ID NO 29 <211> LENGTH: 2295 <212> TYPE: DNA<213> ORGANISM: Saccharomyces cerevisiae <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)...(2295) <400> SEQUENCE: 29 atg tca aca aac tcattc cat gat tat gtg gat tta aaa tcg aga act 48 Met Ser Thr Asn Ser PheHis Asp Tyr Val Asp Leu Lys Ser Arg Thr 1 5 10 15 aat aca cga cag ttttca gat gac gaa gag ttc act acg cct cca aaa 96 Asn Thr Arg Gln Phe SerAsp Asp Glu Glu Phe Thr Thr Pro Pro Lys 20 25 30 cta agc aat ttc gga tcagct tta ctt tcc cac aca gaa aaa act tca 144 Leu Ser Asn Phe Gly Ser AlaLeu Leu Ser His Thr Glu Lys Thr Ser 35 40 45 gca tca gag ata tta tca agtcat aat aat gac aag atc gca aat cga 192 Ala Ser Glu Ile Leu Ser Ser HisAsn Asn Asp Lys Ile Ala Asn Arg 50 55 60 tta gaa gaa atg gac agg agt tcatca agg agt cac ccc cca ccg tca 240 Leu Glu Glu Met Asp Arg Ser Ser SerArg Ser His Pro Pro Pro Ser 65 70 75 80 atg ggc aat ttg aca tcc ggt catact agt acc tca tcg cat tca acc 288 Met Gly Asn Leu Thr Ser Gly His ThrSer Thr Ser Ser His Ser Thr 85 90 95 ttg ttc gga cga tat ctg aga aat aatcac cag act agc atg acg acg 336 Leu Phe Gly Arg Tyr Leu Arg Asn Asn HisGln Thr Ser Met Thr Thr 100 105 110 atg aac act agt gac ata gag ata aatgtt gga aat agt ctt gat aag 384 Met Asn Thr Ser Asp Ile Glu Ile Asn ValGly Asn Ser Leu Asp Lys 115 120 125 agt ttt gaa agg ata agg aat ttg cgacaa aat atg aaa gaa gat att 432 Ser Phe Glu Arg Ile Arg Asn Leu Arg GlnAsn Met Lys Glu Asp Ile 130 135 140 acg gca aag tat gct gaa agg aga agtaag aga ttt tta ata tcc aat 480 Thr Ala Lys Tyr Ala Glu Arg Arg Ser LysArg Phe Leu Ile Ser Asn 145 150 155 160 agg aca acg aag ctg ggt cct gcaaag aga gcg atg act ttg aca aat 528 Arg Thr Thr Lys Leu Gly Pro Ala LysArg Ala Met Thr Leu Thr Asn 165 170 175 atc ttt gat gag gat gtg cct aactct cca aac cag cca ata aat gca 576 Ile Phe Asp Glu Asp Val Pro Asn SerPro Asn Gln Pro Ile Asn Ala 180 185 190 agg gag aca gtg gaa tta cca cttgag gat tct cac caa aca aac ttt 624 Arg Glu Thr Val Glu Leu Pro Leu GluAsp Ser His Gln Thr Asn Phe 195 200 205 aaa gaa cga aga gag aat acg gattat gat tca att gat ttt gga gat 672 Lys Glu Arg Arg Glu Asn Thr Asp TyrAsp Ser Ile Asp Phe Gly Asp 210 215 220 ttg aat cct atc cag tat att aaaaaa cat aat ctt ccc aca agt gac 720 Leu Asn Pro Ile Gln Tyr Ile Lys LysHis Asn Leu Pro Thr Ser Asp 225 230 235 240 ctt cca cta ata tct caa atctac ttt gat aaa caa aga gaa gaa aat 768 Leu Pro Leu Ile Ser Gln Ile TyrPhe Asp Lys Gln Arg Glu Glu Asn 245 250 255 aga caa gca gca ctc cga aaacat agt tcc aga gaa ttg ctt tat aaa 816 Arg Gln Ala Ala Leu Arg Lys HisSer Ser Arg Glu Leu Leu Tyr Lys 260 265 270 agt agg tct tct tcc tct tcactt tct agt aac aac tta ttg gca aac 864 Ser Arg Ser Ser Ser Ser Ser LeuSer Ser Asn Asn Leu Leu Ala Asn 275 280 285 aag gac aat tct ata aca tccaat aat ggt tct caa ccc agg cga aaa 912 Lys Asp Asn Ser Ile Thr Ser AsnAsn Gly Ser Gln Pro Arg Arg Lys 290 295 300 gtt tct act gga tca tct tcatct aag tca tcg atc gaa ata aga aga 960 Val Ser Thr Gly Ser Ser Ser SerLys Ser Ser Ile Glu Ile Arg Arg 305 310 315 320 gct ctc aag gag aat attgat act agc aat aac agc aat ttc aac agc 1008 Ala Leu Lys Glu Asn Ile AspThr Ser Asn Asn Ser Asn Phe Asn Ser 325 330 335 cca att cat aaa att tataaa gga att tcc aga aat aaa gat tcc gac 1056 Pro Ile His Lys Ile Tyr LysGly Ile Ser Arg Asn Lys Asp Ser Asp 340 345 350 tcc gaa aaa aga gaa gtactg cga aac ata agc ata aat gca aat cac 1104 Ser Glu Lys Arg Glu Val LeuArg Asn Ile Ser Ile Asn Ala Asn His 355 360 365 gct gat aat ctc ctt caacaa gag aat aag aga cta aaa cga tca ttg 1152 Ala Asp Asn Leu Leu Gln GlnGlu Asn Lys Arg Leu Lys Arg Ser Leu 370 375 380 gat gat gca ata acg aatgag aat ata aac agt aaa aat cta gaa gta 1200 Asp Asp Ala Ile Thr Asn GluAsn Ile Asn Ser Lys Asn Leu Glu Val 385 390 395 400 ttt tac cat cga cctgct ccc aaa cct cca gtc acc aag aaa gtt gaa 1248 Phe Tyr His Arg Pro AlaPro Lys Pro Pro Val Thr Lys Lys Val Glu 405 410 415 att gtt gaa cct gcaaag tcc gct tct tta tcg aat aat aga aat ata 1296 Ile Val Glu Pro Ala LysSer Ala Ser Leu Ser Asn Asn Arg Asn Ile 420 425 430 att aca gta aat gactcc cag tac gaa aaa ata gaa ctt ttg ggt aga 1344 Ile Thr Val Asn Asp SerGln Tyr Glu Lys Ile Glu Leu Leu Gly Arg 435 440 445 ggt gga tcc tcc agagtt tac aag gtg aaa gga tct ggc aat agg gta 1392 Gly Gly Ser Ser Arg ValTyr Lys Val Lys Gly Ser Gly Asn Arg Val 450 455 460 tac gcg ctc aaa agggtg tct ttt gac gct ttt gac gat tca agt att 1440 Tyr Ala Leu Lys Arg ValSer Phe Asp Ala Phe Asp Asp Ser Ser Ile 465 470 475 480 gat gga ttc aaagga gaa ata gaa ctt ctg gaa aaa ttg aaa gac caa 1488 Asp Gly Phe Lys GlyGlu Ile Glu Leu Leu Glu Lys Leu Lys Asp Gln 485 490 495 aag cgt gta atccaa cta cta gat tat gaa atg ggg gat ggt tta ttg 1536 Lys Arg Val Ile GlnLeu Leu Asp Tyr Glu Met Gly Asp Gly Leu Leu 500 505 510 tat ttg ata atggaa tgt ggt gat cat gat ttg tca caa atc ctt aac 1584 Tyr Leu Ile Met GluCys Gly Asp His Asp Leu Ser Gln Ile Leu Asn 515 520 525 caa aga agc ggcatg cca ctg gat ttt aat ttt gtt aga ttc tat aca 1632 Gln Arg Ser Gly MetPro Leu Asp Phe Asn Phe Val Arg Phe Tyr Thr 530 535 540 aag gaa atg ttgctg tgc att aaa gta gtt cat gat gcg ggc ata gtt 1680 Lys Glu Met Leu LeuCys Ile Lys Val Val His Asp Ala Gly Ile Val 545 550 555 560 cat tcg gattta aaa cct gca aat ttt gtt tta gtg aaa ggt atc tta 1728 His Ser Asp LeuLys Pro Ala Asn Phe Val Leu Val Lys Gly Ile Leu 565 570 575 aaa atc attgat ttt ggt ata gca aac gcg gta ccg gaa cat acg gtg 1776 Lys Ile Ile AspPhe Gly Ile Ala Asn Ala Val Pro Glu His Thr Val 580 585 590 aat ata tatcgt gaa act caa att ggg act cca aat tat atg gca cca 1824 Asn Ile Tyr ArgGlu Thr Gln Ile Gly Thr Pro Asn Tyr Met Ala Pro 595 600 605 gaa gca ctagtt gct atg aat tac aca caa aat agt gag aac caa cat 1872 Glu Ala Leu ValAla Met Asn Tyr Thr Gln Asn Ser Glu Asn Gln His 610 615 620 gag gga aacaag tgg aaa gtg ggg aga cca tct gat atg tgg tca tgc 1920 Glu Gly Asn LysTrp Lys Val Gly Arg Pro Ser Asp Met Trp Ser Cys 625 630 635 640 ggt tgtatt ata tat cag atg att tac ggg aaa ccc cca tat ggc agt 1968 Gly Cys IleIle Tyr Gln Met Ile Tyr Gly Lys Pro Pro Tyr Gly Ser 645 650 655 ttc caaggc caa aat agg ctg ttg gct att atg aat cct gat gtg aaa 2016 Phe Gln GlyGln Asn Arg Leu Leu Ala Ile Met Asn Pro Asp Val Lys 660 665 670 atc ccattt cct gaa cat act agc aat aat gaa aag att cca aag tct 2064 Ile Pro PhePro Glu His Thr Ser Asn Asn Glu Lys Ile Pro Lys Ser 675 680 685 gcc attgaa tta atg aaa gca tgt ctg tac agg aac cca gac aaa aga 2112 Ala Ile GluLeu Met Lys Ala Cys Leu Tyr Arg Asn Pro Asp Lys Arg 690 695 700 tgg actgtg gat aaa gtc ctg agt agc act ttc ctt caa cct ttt atg 2160 Trp Thr ValAsp Lys Val Leu Ser Ser Thr Phe Leu Gln Pro Phe Met 705 710 715 720 atatcc gga tcg att atg gaa gac ctt att agg aat gcc gtt aga tat 2208 Ile SerGly Ser Ile Met Glu Asp Leu Ile Arg Asn Ala Val Arg Tyr 725 730 735 ggctct gag aag cct cat ata tca caa gat gat ctc aat gat gtg gta 2256 Gly SerGlu Lys Pro His Ile Ser Gln Asp Asp Leu Asn Asp Val Val 740 745 750 gacact gtt tta agg aaa ttt gca gat tac aaa att tag 2295 Asp Thr Val Leu ArgLys Phe Ala Asp Tyr Lys Ile * 755 760 <210> SEQ ID NO 30 <211> LENGTH:764 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400>SEQUENCE: 30 Met Ser Thr Asn Ser Phe His Asp Tyr Val Asp Leu Lys Ser ArgThr 1 5 10 15 Asn Thr Arg Gln Phe Ser Asp Asp Glu Glu Phe Thr Thr ProPro Lys 20 25 30 Leu Ser Asn Phe Gly Ser Ala Leu Leu Ser His Thr Glu LysThr Ser 35 40 45 Ala Ser Glu Ile Leu Ser Ser His Asn Asn Asp Lys Ile AlaAsn Arg 50 55 60 Leu Glu Glu Met Asp Arg Ser Ser Ser Arg Ser His Pro ProPro Ser 65 70 75 80 Met Gly Asn Leu Thr Ser Gly His Thr Ser Thr Ser SerHis Ser Thr 85 90 95 Leu Phe Gly Arg Tyr Leu Arg Asn Asn His Gln Thr SerMet Thr Thr 100 105 110 Met Asn Thr Ser Asp Ile Glu Ile Asn Val Gly AsnSer Leu Asp Lys 115 120 125 Ser Phe Glu Arg Ile Arg Asn Leu Arg Gln AsnMet Lys Glu Asp Ile 130 135 140 Thr Ala Lys Tyr Ala Glu Arg Arg Ser LysArg Phe Leu Ile Ser Asn 145 150 155 160 Arg Thr Thr Lys Leu Gly Pro AlaLys Arg Ala Met Thr Leu Thr Asn 165 170 175 Ile Phe Asp Glu Asp Val ProAsn Ser Pro Asn Gln Pro Ile Asn Ala 180 185 190 Arg Glu Thr Val Glu LeuPro Leu Glu Asp Ser His Gln Thr Asn Phe 195 200 205 Lys Glu Arg Arg GluAsn Thr Asp Tyr Asp Ser Ile Asp Phe Gly Asp 210 215 220 Leu Asn Pro IleGln Tyr Ile Lys Lys His Asn Leu Pro Thr Ser Asp 225 230 235 240 Leu ProLeu Ile Ser Gln Ile Tyr Phe Asp Lys Gln Arg Glu Glu Asn 245 250 255 ArgGln Ala Ala Leu Arg Lys His Ser Ser Arg Glu Leu Leu Tyr Lys 260 265 270Ser Arg Ser Ser Ser Ser Ser Leu Ser Ser Asn Asn Leu Leu Ala Asn 275 280285 Lys Asp Asn Ser Ile Thr Ser Asn Asn Gly Ser Gln Pro Arg Arg Lys 290295 300 Val Ser Thr Gly Ser Ser Ser Ser Lys Ser Ser Ile Glu Ile Arg Arg305 310 315 320 Ala Leu Lys Glu Asn Ile Asp Thr Ser Asn Asn Ser Asn PheAsn Ser 325 330 335 Pro Ile His Lys Ile Tyr Lys Gly Ile Ser Arg Asn LysAsp Ser Asp 340 345 350 Ser Glu Lys Arg Glu Val Leu Arg Asn Ile Ser IleAsn Ala Asn His 355 360 365 Ala Asp Asn Leu Leu Gln Gln Glu Asn Lys ArgLeu Lys Arg Ser Leu 370 375 380 Asp Asp Ala Ile Thr Asn Glu Asn Ile AsnSer Lys Asn Leu Glu Val 385 390 395 400 Phe Tyr His Arg Pro Ala Pro LysPro Pro Val Thr Lys Lys Val Glu 405 410 415 Ile Val Glu Pro Ala Lys SerAla Ser Leu Ser Asn Asn Arg Asn Ile 420 425 430 Ile Thr Val Asn Asp SerGln Tyr Glu Lys Ile Glu Leu Leu Gly Arg 435 440 445 Gly Gly Ser Ser ArgVal Tyr Lys Val Lys Gly Ser Gly Asn Arg Val 450 455 460 Tyr Ala Leu LysArg Val Ser Phe Asp Ala Phe Asp Asp Ser Ser Ile 465 470 475 480 Asp GlyPhe Lys Gly Glu Ile Glu Leu Leu Glu Lys Leu Lys Asp Gln 485 490 495 LysArg Val Ile Gln Leu Leu Asp Tyr Glu Met Gly Asp Gly Leu Leu 500 505 510Tyr Leu Ile Met Glu Cys Gly Asp His Asp Leu Ser Gln Ile Leu Asn 515 520525 Gln Arg Ser Gly Met Pro Leu Asp Phe Asn Phe Val Arg Phe Tyr Thr 530535 540 Lys Glu Met Leu Leu Cys Ile Lys Val Val His Asp Ala Gly Ile Val545 550 555 560 His Ser Asp Leu Lys Pro Ala Asn Phe Val Leu Val Lys GlyIle Leu 565 570 575 Lys Ile Ile Asp Phe Gly Ile Ala Asn Ala Val Pro GluHis Thr Val 580 585 590 Asn Ile Tyr Arg Glu Thr Gln Ile Gly Thr Pro AsnTyr Met Ala Pro 595 600 605 Glu Ala Leu Val Ala Met Asn Tyr Thr Gln AsnSer Glu Asn Gln His 610 615 620 Glu Gly Asn Lys Trp Lys Val Gly Arg ProSer Asp Met Trp Ser Cys 625 630 635 640 Gly Cys Ile Ile Tyr Gln Met IleTyr Gly Lys Pro Pro Tyr Gly Ser 645 650 655 Phe Gln Gly Gln Asn Arg LeuLeu Ala Ile Met Asn Pro Asp Val Lys 660 665 670 Ile Pro Phe Pro Glu HisThr Ser Asn Asn Glu Lys Ile Pro Lys Ser 675 680 685 Ala Ile Glu Leu MetLys Ala Cys Leu Tyr Arg Asn Pro Asp Lys Arg 690 695 700 Trp Thr Val AspLys Val Leu Ser Ser Thr Phe Leu Gln Pro Phe Met 705 710 715 720 Ile SerGly Ser Ile Met Glu Asp Leu Ile Arg Asn Ala Val Arg Tyr 725 730 735 GlySer Glu Lys Pro His Ile Ser Gln Asp Asp Leu Asn Asp Val Val 740 745 750Asp Thr Val Leu Arg Lys Phe Ala Asp Tyr Lys Ile 755 760 <210> SEQ ID NO31 <211> LENGTH: 2079 <212> TYPE: DNA <213> ORGANISM:Schizosaccharomyces pombe <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (30)...(2066) <400> SEQUENCE: 31 ggaatgctcg ttttttagtaactgtgttt atg tct aag cgc aat cct cct gtg 53 Met Ser Lys Arg Asn Pro ProVal 1 5 act aat atc gcg gac ttg gtg tca gat tct tcc tta gat gaa gac tcg101 Thr Asn Ile Ala Asp Leu Val Ser Asp Ser Ser Leu Asp Glu Asp Ser 1015 20 ctt tct ttt ctc gaa gag ctt cag gat cca gaa tta tac ttc aaa aac149 Leu Ser Phe Leu Glu Glu Leu Gln Asp Pro Glu Leu Tyr Phe Lys Asn 2530 35 40 gac act ttc tct tcc aag agt agc cat tct gat ggc acc gtt act ggg197 Asp Thr Phe Ser Ser Lys Ser Ser His Ser Asp Gly Thr Val Thr Gly 4550 55 gat acg ttg cgt agg cag tca agc ggt gca act gct tta gag aga ttg245 Asp Thr Leu Arg Arg Gln Ser Ser Gly Ala Thr Ala Leu Glu Arg Leu 6065 70 gtc tca cat cct cgt act aaa aat ttt gat ttg caa gga aat gga gga293 Val Ser His Pro Arg Thr Lys Asn Phe Asp Leu Gln Gly Asn Gly Gly 7580 85 caa aat tct gct ttg aag gaa gtg aat act cca gca tat cag tca atg341 Gln Asn Ser Ala Leu Lys Glu Val Asn Thr Pro Ala Tyr Gln Ser Met 9095 100 cac cat ttc gag cat tta ata aca ccc ttg ccc tct act aat gcg tct389 His His Phe Glu His Leu Ile Thr Pro Leu Pro Ser Thr Asn Ala Ser 105110 115 120 cac agt gaa gtt tca ctc agt gca gga gtg aat gat ctc aat tctaat 437 His Ser Glu Val Ser Leu Ser Ala Gly Val Asn Asp Leu Asn Ser Asn125 130 135 tcg gag cat gat ttg tta cct aaa agt gta aac aaa acc ccc ggttct 485 Ser Glu His Asp Leu Leu Pro Lys Ser Val Asn Lys Thr Pro Gly Ser140 145 150 tta tca att tca aga cga cga aga atc ggc aga att gga tta ggccct 533 Leu Ser Ile Ser Arg Arg Arg Arg Ile Gly Arg Ile Gly Leu Gly Pro155 160 165 cca aag cgt gct gag tac acg ttg acg gat ccc tcg aag act tccgat 581 Pro Lys Arg Ala Glu Tyr Thr Leu Thr Asp Pro Ser Lys Thr Ser Asp170 175 180 acc aaa aac tct aca gaa gca gat gag gat att gaa atg aaa tctcga 629 Thr Lys Asn Ser Thr Glu Ala Asp Glu Asp Ile Glu Met Lys Ser Arg185 190 195 200 gaa gta tca cca gct tcc aac tct gtt gct gca aca act ttaaaa cct 677 Glu Val Ser Pro Ala Ser Asn Ser Val Ala Ala Thr Thr Leu LysPro 205 210 215 ctg cag ctg cat aac act cct ttg caa aca tcc cag gag catccc aaa 725 Leu Gln Leu His Asn Thr Pro Leu Gln Thr Ser Gln Glu His ProLys 220 225 230 cct tct ttt cat cct tct cag ttt gag agc tct ttt tct cctagg gtg 773 Pro Ser Phe His Pro Ser Gln Phe Glu Ser Ser Phe Ser Pro ArgVal 235 240 245 cag ttt gat cac gat gtt gaa aga aga gct agt gaa ctt cattct cgt 821 Gln Phe Asp His Asp Val Glu Arg Arg Ala Ser Glu Leu His SerArg 250 255 260 cca gtc acc gtt ttc caa gag cct cag cgt tct gct tct caacca tat 869 Pro Val Thr Val Phe Gln Glu Pro Gln Arg Ser Ala Ser Gln ProTyr 265 270 275 280 gaa tct cat gct ctt tct cca aag gtg gct ccg tta tttgat aac agt 917 Glu Ser His Ala Leu Ser Pro Lys Val Ala Pro Leu Phe AspAsn Ser 285 290 295 caa gct act ccc ata ccc aag cgt cag cag gac gtt gttact gtt gcc 965 Gln Ala Thr Pro Ile Pro Lys Arg Gln Gln Asp Val Val ThrVal Ala 300 305 310 aat cta caa ttt atc aaa tta gga gtt gtt gga aag ggtgga agt agt 1013 Asn Leu Gln Phe Ile Lys Leu Gly Val Val Gly Lys Gly GlySer Ser 315 320 325 atg gta tat cgc ata ttt tcc ccc gat aac agt cgt ttatac gct ttg 1061 Met Val Tyr Arg Ile Phe Ser Pro Asp Asn Ser Arg Leu TyrAla Leu 330 335 340 aaa gag gtg aac ttt att aat gca gac caa act act atacaa gga tac 1109 Lys Glu Val Asn Phe Ile Asn Ala Asp Gln Thr Thr Ile GlnGly Tyr 345 350 355 360 aag aac gaa att gca tta tta aga aag ctt tca ggcaat gat cgc ata 1157 Lys Asn Glu Ile Ala Leu Leu Arg Lys Leu Ser Gly AsnAsp Arg Ile 365 370 375 att aaa tta tat gct gcc gaa gtt aat gat act ttaggg caa ctc aat 1205 Ile Lys Leu Tyr Ala Ala Glu Val Asn Asp Thr Leu GlyGln Leu Asn 380 385 390 atg gtg atg gaa tgc gga gaa acg gat tta gca aacctt tta atg aaa 1253 Met Val Met Glu Cys Gly Glu Thr Asp Leu Ala Asn LeuLeu Met Lys 395 400 405 aac atg aag aaa ccc att aat ctt aat ttc atc agaatg tat tgg gag 1301 Asn Met Lys Lys Pro Ile Asn Leu Asn Phe Ile Arg MetTyr Trp Glu 410 415 420 caa atg cta gag gcg gtc cag gta gtt cat gat caaaat ata gtg cat 1349 Gln Met Leu Glu Ala Val Gln Val Val His Asp Gln AsnIle Val His 425 430 435 440 tcg gat ttg aag ccg gcc aat ttc ctg ctt gtagaa ggg aat ttg aag 1397 Ser Asp Leu Lys Pro Ala Asn Phe Leu Leu Val GluGly Asn Leu Lys 445 450 455 ctg att gat ttt ggc att gcc aaa gca att ggtaat gac acc act aat 1445 Leu Ile Asp Phe Gly Ile Ala Lys Ala Ile Gly AsnAsp Thr Thr Asn 460 465 470 atc cat cgt gat tcc cac atc ggt act att aattat atg gca cct gaa 1493 Ile His Arg Asp Ser His Ile Gly Thr Ile Asn TyrMet Ala Pro Glu 475 480 485 gct ttg aca gac atg aat gct cac aca aac tctggc gtg aaa ctc gta 1541 Ala Leu Thr Asp Met Asn Ala His Thr Asn Ser GlyVal Lys Leu Val 490 495 500 aag ttg ggc agg ccc agc gac gtg tgg agt ttggga tgt ata tta tat 1589 Lys Leu Gly Arg Pro Ser Asp Val Trp Ser Leu GlyCys Ile Leu Tyr 505 510 515 520 cag atg gtg tat ggg agg gcc ccg ttt gctcat cta aaa atg atc caa 1637 Gln Met Val Tyr Gly Arg Ala Pro Phe Ala HisLeu Lys Met Ile Gln 525 530 535 gct ata gca gct atc cct aat gaa caa tatcac att cat ttc ccc gaa 1685 Ala Ile Ala Ala Ile Pro Asn Glu Gln Tyr HisIle His Phe Pro Glu 540 545 550 gtt gcc tta cct gct aat gct gtc cag gagaaa gag gga tcg ttg cca 1733 Val Ala Leu Pro Ala Asn Ala Val Gln Glu LysGlu Gly Ser Leu Pro 555 560 565 ggt gta act gtc ggg cct gat cta atg gatgtt atg aaa aga tgc ctg 1781 Gly Val Thr Val Gly Pro Asp Leu Met Asp ValMet Lys Arg Cys Leu 570 575 580 gaa agg gat caa cgg aag aga ctt aca ataccg gaa ttg ctg gtt cat 1829 Glu Arg Asp Gln Arg Lys Arg Leu Thr Ile ProGlu Leu Leu Val His 585 590 595 600 ccc ttt tta aac cct ttg cca tcc tatttg aca cct ttg gcc aaa aag 1877 Pro Phe Leu Asn Pro Leu Pro Ser Tyr LeuThr Pro Leu Ala Lys Lys 605 610 615 ccg tta cct gtt tct ggg cac acc aataat gct cat cca ctt aga ctc 1925 Pro Leu Pro Val Ser Gly His Thr Asn AsnAla His Pro Leu Arg Leu 620 625 630 agc aca gaa atc tca gct tct caa ttatca atg att ata gaa agg tcg 1973 Ser Thr Glu Ile Ser Ala Ser Gln Leu SerMet Ile Ile Glu Arg Ser 635 640 645 gtg gag ttg agt aag cac aag cga ttaaat aag gaa ctt att gat agc 2021 Val Glu Leu Ser Lys His Lys Arg Leu AsnLys Glu Leu Ile Asp Ser 650 655 660 atg gct tat gat tgc gtt agc aat ttacga aaa atg cca gaa tag 2066 Met Ala Tyr Asp Cys Val Ser Asn Leu Arg LysMet Pro Glu * 665 670 675 aggcactaaa ttt 2079 <210> SEQ ID NO 32 <211>LENGTH: 678 <212> TYPE: PRT <213> ORGANISM: Schizosaccharomyces pombe<400> SEQUENCE: 32 Met Ser Lys Arg Asn Pro Pro Val Thr Asn Ile Ala AspLeu Val Ser 1 5 10 15 Asp Ser Ser Leu Asp Glu Asp Ser Leu Ser Phe LeuGlu Glu Leu Gln 20 25 30 Asp Pro Glu Leu Tyr Phe Lys Asn Asp Thr Phe SerSer Lys Ser Ser 35 40 45 His Ser Asp Gly Thr Val Thr Gly Asp Thr Leu ArgArg Gln Ser Ser 50 55 60 Gly Ala Thr Ala Leu Glu Arg Leu Val Ser His ProArg Thr Lys Asn 65 70 75 80 Phe Asp Leu Gln Gly Asn Gly Gly Gln Asn SerAla Leu Lys Glu Val 85 90 95 Asn Thr Pro Ala Tyr Gln Ser Met His His PheGlu His Leu Ile Thr 100 105 110 Pro Leu Pro Ser Thr Asn Ala Ser His SerGlu Val Ser Leu Ser Ala 115 120 125 Gly Val Asn Asp Leu Asn Ser Asn SerGlu His Asp Leu Leu Pro Lys 130 135 140 Ser Val Asn Lys Thr Pro Gly SerLeu Ser Ile Ser Arg Arg Arg Arg 145 150 155 160 Ile Gly Arg Ile Gly LeuGly Pro Pro Lys Arg Ala Glu Tyr Thr Leu 165 170 175 Thr Asp Pro Ser LysThr Ser Asp Thr Lys Asn Ser Thr Glu Ala Asp 180 185 190 Glu Asp Ile GluMet Lys Ser Arg Glu Val Ser Pro Ala Ser Asn Ser 195 200 205 Val Ala AlaThr Thr Leu Lys Pro Leu Gln Leu His Asn Thr Pro Leu 210 215 220 Gln ThrSer Gln Glu His Pro Lys Pro Ser Phe His Pro Ser Gln Phe 225 230 235 240Glu Ser Ser Phe Ser Pro Arg Val Gln Phe Asp His Asp Val Glu Arg 245 250255 Arg Ala Ser Glu Leu His Ser Arg Pro Val Thr Val Phe Gln Glu Pro 260265 270 Gln Arg Ser Ala Ser Gln Pro Tyr Glu Ser His Ala Leu Ser Pro Lys275 280 285 Val Ala Pro Leu Phe Asp Asn Ser Gln Ala Thr Pro Ile Pro LysArg 290 295 300 Gln Gln Asp Val Val Thr Val Ala Asn Leu Gln Phe Ile LysLeu Gly 305 310 315 320 Val Val Gly Lys Gly Gly Ser Ser Met Val Tyr ArgIle Phe Ser Pro 325 330 335 Asp Asn Ser Arg Leu Tyr Ala Leu Lys Glu ValAsn Phe Ile Asn Ala 340 345 350 Asp Gln Thr Thr Ile Gln Gly Tyr Lys AsnGlu Ile Ala Leu Leu Arg 355 360 365 Lys Leu Ser Gly Asn Asp Arg Ile IleLys Leu Tyr Ala Ala Glu Val 370 375 380 Asn Asp Thr Leu Gly Gln Leu AsnMet Val Met Glu Cys Gly Glu Thr 385 390 395 400 Asp Leu Ala Asn Leu LeuMet Lys Asn Met Lys Lys Pro Ile Asn Leu 405 410 415 Asn Phe Ile Arg MetTyr Trp Glu Gln Met Leu Glu Ala Val Gln Val 420 425 430 Val His Asp GlnAsn Ile Val His Ser Asp Leu Lys Pro Ala Asn Phe 435 440 445 Leu Leu ValGlu Gly Asn Leu Lys Leu Ile Asp Phe Gly Ile Ala Lys 450 455 460 Ala IleGly Asn Asp Thr Thr Asn Ile His Arg Asp Ser His Ile Gly 465 470 475 480Thr Ile Asn Tyr Met Ala Pro Glu Ala Leu Thr Asp Met Asn Ala His 485 490495 Thr Asn Ser Gly Val Lys Leu Val Lys Leu Gly Arg Pro Ser Asp Val 500505 510 Trp Ser Leu Gly Cys Ile Leu Tyr Gln Met Val Tyr Gly Arg Ala Pro515 520 525 Phe Ala His Leu Lys Met Ile Gln Ala Ile Ala Ala Ile Pro AsnGlu 530 535 540 Gln Tyr His Ile His Phe Pro Glu Val Ala Leu Pro Ala AsnAla Val 545 550 555 560 Gln Glu Lys Glu Gly Ser Leu Pro Gly Val Thr ValGly Pro Asp Leu 565 570 575 Met Asp Val Met Lys Arg Cys Leu Glu Arg AspGln Arg Lys Arg Leu 580 585 590 Thr Ile Pro Glu Leu Leu Val His Pro PheLeu Asn Pro Leu Pro Ser 595 600 605 Tyr Leu Thr Pro Leu Ala Lys Lys ProLeu Pro Val Ser Gly His Thr 610 615 620 Asn Asn Ala His Pro Leu Arg LeuSer Thr Glu Ile Ser Ala Ser Gln 625 630 635 640 Leu Ser Met Ile Ile GluArg Ser Val Glu Leu Ser Lys His Lys Arg 645 650 655 Leu Asn Lys Glu LeuIle Asp Ser Met Ala Tyr Asp Cys Val Ser Asn 660 665 670 Leu Arg Lys MetPro Glu 675 <210> SEQ ID NO 33 <211> LENGTH: 2263 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (379)...(1491) <400> SEQUENCE: 33 gatatcacag caacattgaaatgctaaaaa gtttttaaac actctcaatt tctaattcac 60 catgtcacag actggtgaaaaaaaaaaaaa aagcggccgc ttccccccgg ccgggccccc 120 gccgccccgc ggtccccagagcgccaggcc cccgggggga gggagggagg gcgccgggcc 180 ggtgggagcc agcggcgcgcggtgggaccc acggagcccc gcgacccgcc gagcctggag 240 ccgggccggc tcggggaagccggctccagc ccggagcgaa cttcgcagcc cgtcgggggg 300 cggcggggag ggggcccggagccggaggag ggggcggccg cgggcacccc cgcctgtgcc 360 ccggcgtccc cgggcacc atgctg tcc aac tcc cag ggc cag agc ccg ccg 411 Met Leu Ser Asn Ser Gln GlyGln Ser Pro Pro 1 5 10 gtg ccg ttc ccc gcc ccg gcc ccg ccg ccg cag cccccc acc cct gcc 459 Val Pro Phe Pro Ala Pro Ala Pro Pro Pro Gln Pro ProThr Pro Ala 15 20 25 ctg ccg cac ccc ccg gcg cag ccg ccg ccg ccg ccc ccgcag cag ttc 507 Leu Pro His Pro Pro Ala Gln Pro Pro Pro Pro Pro Pro GlnGln Phe 30 35 40 ccg cag ttc cac gtc aag tcc ggc ctg cag atc aag aag aacgcc atc 555 Pro Gln Phe His Val Lys Ser Gly Leu Gln Ile Lys Lys Asn AlaIle 45 50 55 atc gat gac tac aag gtc acc agc cag gtc ctg ggg ctg ggc atcaac 603 Ile Asp Asp Tyr Lys Val Thr Ser Gln Val Leu Gly Leu Gly Ile Asn60 65 70 75 ggc aaa gtt ttg cag atc ttc aac aag agg acc cag gag aaa ttcgcc 651 Gly Lys Val Leu Gln Ile Phe Asn Lys Arg Thr Gln Glu Lys Phe Ala80 85 90 ctc aaa atg ctt cag gac tgc ccc aag gcc cgc agg gag gtg gag ctg699 Leu Lys Met Leu Gln Asp Cys Pro Lys Ala Arg Arg Glu Val Glu Leu 95100 105 cac tgg cgg gcc tcc cag tgc ccg cac atc gta cgg atc gtg gat gtg747 His Trp Arg Ala Ser Gln Cys Pro His Ile Val Arg Ile Val Asp Val 110115 120 tac gag aat ctg tac gca ggg agg aag tgc ctg ctg att gtc atg gaa795 Tyr Glu Asn Leu Tyr Ala Gly Arg Lys Cys Leu Leu Ile Val Met Glu 125130 135 tgt ttg gac ggt gga gaa ctc ttt agc cga atc cag gat cga gga gac843 Cys Leu Asp Gly Gly Glu Leu Phe Ser Arg Ile Gln Asp Arg Gly Asp 140145 150 155 cag gca ttc aca gaa aga gaa gca tcc gaa atc atg aag agc atcggt 891 Gln Ala Phe Thr Glu Arg Glu Ala Ser Glu Ile Met Lys Ser Ile Gly160 165 170 gag gcc atc cag tat ctg cat tca atc aac att gcc cat cgg gatgtc 939 Glu Ala Ile Gln Tyr Leu His Ser Ile Asn Ile Ala His Arg Asp Val175 180 185 aag cct gag aat ctc tta tac acc tcc aaa agg ccc aac gcc atcctg 987 Lys Pro Glu Asn Leu Leu Tyr Thr Ser Lys Arg Pro Asn Ala Ile Leu190 195 200 aaa ctc act gac ttt ggc ttt gcc aag gaa acc acc agc cac aactct 1035 Lys Leu Thr Asp Phe Gly Phe Ala Lys Glu Thr Thr Ser His Asn Ser205 210 215 ttg acc act cct tgt tat aca ccg tac tat gtg gct cca gaa gtgctg 1083 Leu Thr Thr Pro Cys Tyr Thr Pro Tyr Tyr Val Ala Pro Glu Val Leu220 225 230 235 ggt cca gag aag tat gac aag tcc tgt gac atg tgg tcc ctgggt gtc 1131 Gly Pro Glu Lys Tyr Asp Lys Ser Cys Asp Met Trp Ser Leu GlyVal 240 245 250 atc atg tac atc ctg ctg tgt ggg tat ccc ccc ttc tac tccaac cac 1179 Ile Met Tyr Ile Leu Leu Cys Gly Tyr Pro Pro Phe Tyr Ser AsnHis 255 260 265 ggc ctt gcc atc tct ccg ggc atg aag act cgc atc cga atgggc cag 1227 Gly Leu Ala Ile Ser Pro Gly Met Lys Thr Arg Ile Arg Met GlyGln 270 275 280 tat gaa ttt ccc aac cca gaa tgg tca gaa gta tca gag gaagtg aag 1275 Tyr Glu Phe Pro Asn Pro Glu Trp Ser Glu Val Ser Glu Glu ValLys 285 290 295 atg ctc att cgg aat ctg ctg aaa aca gag ccc acc cag agaatg acc 1323 Met Leu Ile Arg Asn Leu Leu Lys Thr Glu Pro Thr Gln Arg MetThr 300 305 310 315 atc acc gag ttt atg aac cac cct tgg atc atg caa tcaaca aag gtc 1371 Ile Thr Glu Phe Met Asn His Pro Trp Ile Met Gln Ser ThrLys Val 320 325 330 cct caa acc cca ctg cac acc agc cgg gtc ctg aag gaggac aag gag 1419 Pro Gln Thr Pro Leu His Thr Ser Arg Val Leu Lys Glu AspLys Glu 335 340 345 cgg tgg gag gat gtc aag ggg tgt ctt cat gac aag aacagc gac cag 1467 Arg Trp Glu Asp Val Lys Gly Cys Leu His Asp Lys Asn SerAsp Gln 350 355 360 gcc act tgg ctg acc agg ttg tga gcagaggattctgtgttcct gtccaaactc 1521 Ala Thr Trp Leu Thr Arg Leu * 365 370agtgctgttt cttagaatcc ttttattccc tgggtctcta atgggacctt aaagaccatc 1581tggtatcatc ttctcatttt gcagaagaga aactgaggcc cagaggcgga gggcagtctg 1641ctcaaggtca cgcagctggt gactggttgg ggcagaccgg acccaggttt cctgactcct 1701ggcccaagtc tcttcctcct atcctgcggg atcactgggg ggctctcagg gaacagcagc 1761agtgccatag ccaggctctc tgctgcccag cgctggggtg aggctgccgt tgtcagcgtg 1821gaccactaac cagcccgtct tctctctctg ctcccacccc tgccgccctc accctgccct 1881tgttgtctct gtctctcacg tctctcttct gctgtctctc ctacctgtct tctggctctc 1941tctgtaccct tcctggtgct gccgtgcccc caggaggaga tgaccagtgc cttggccaca 2001atgcgcgttg actacgagca gatcaagata aaaaagattg aagatgcatc caaccctctg 2061ctgctgaaga ggcggaagaa agctcgggcc ctggaggctg cggctctggc ccactgagcc 2121accgcgccct cctgcccacg ggaggacaag caataactct ctacaggaat atatttttta 2181aacgaagaga cagaactgtc cacatctgcc tcctctcctc ctcagctgca tggagcctgg 2241aactgcatca gtgactgaat tc 2263 <210> SEQ ID NO 34 <211> LENGTH: 370 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 34 Met Leu SerAsn Ser Gln Gly Gln Ser Pro Pro Val Pro Phe Pro Ala 1 5 10 15 Pro AlaPro Pro Pro Gln Pro Pro Thr Pro Ala Leu Pro His Pro Pro 20 25 30 Ala GlnPro Pro Pro Pro Pro Pro Gln Gln Phe Pro Gln Phe His Val 35 40 45 Lys SerGly Leu Gln Ile Lys Lys Asn Ala Ile Ile Asp Asp Tyr Lys 50 55 60 Val ThrSer Gln Val Leu Gly Leu Gly Ile Asn Gly Lys Val Leu Gln 65 70 75 80 IlePhe Asn Lys Arg Thr Gln Glu Lys Phe Ala Leu Lys Met Leu Gln 85 90 95 AspCys Pro Lys Ala Arg Arg Glu Val Glu Leu His Trp Arg Ala Ser 100 105 110Gln Cys Pro His Ile Val Arg Ile Val Asp Val Tyr Glu Asn Leu Tyr 115 120125 Ala Gly Arg Lys Cys Leu Leu Ile Val Met Glu Cys Leu Asp Gly Gly 130135 140 Glu Leu Phe Ser Arg Ile Gln Asp Arg Gly Asp Gln Ala Phe Thr Glu145 150 155 160 Arg Glu Ala Ser Glu Ile Met Lys Ser Ile Gly Glu Ala IleGln Tyr 165 170 175 Leu His Ser Ile Asn Ile Ala His Arg Asp Val Lys ProGlu Asn Leu 180 185 190 Leu Tyr Thr Ser Lys Arg Pro Asn Ala Ile Leu LysLeu Thr Asp Phe 195 200 205 Gly Phe Ala Lys Glu Thr Thr Ser His Asn SerLeu Thr Thr Pro Cys 210 215 220 Tyr Thr Pro Tyr Tyr Val Ala Pro Glu ValLeu Gly Pro Glu Lys Tyr 225 230 235 240 Asp Lys Ser Cys Asp Met Trp SerLeu Gly Val Ile Met Tyr Ile Leu 245 250 255 Leu Cys Gly Tyr Pro Pro PheTyr Ser Asn His Gly Leu Ala Ile Ser 260 265 270 Pro Gly Met Lys Thr ArgIle Arg Met Gly Gln Tyr Glu Phe Pro Asn 275 280 285 Pro Glu Trp Ser GluVal Ser Glu Glu Val Lys Met Leu Ile Arg Asn 290 295 300 Leu Leu Lys ThrGlu Pro Thr Gln Arg Met Thr Ile Thr Glu Phe Met 305 310 315 320 Asn HisPro Trp Ile Met Gln Ser Thr Lys Val Pro Gln Thr Pro Leu 325 330 335 HisThr Ser Arg Val Leu Lys Glu Asp Lys Glu Arg Trp Glu Asp Val 340 345 350Lys Gly Cys Leu His Asp Lys Asn Ser Asp Gln Ala Thr Trp Leu Thr 355 360365 Arg Leu 370 <210> SEQ ID NO 35 <211> LENGTH: 1074 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (628)...(831) <400> SEQUENCE: 35 ttttactttt tttaagcacaaaattttgtt ttttttctcc cctccccaca gatcccatct 60 caaatcattc tgttaaccaccattccaaca ggtcgaggag agcttaaaca ccttcttcct 120 ctgccttgtt tctatttttttatttttttg catcagtatt aatgtttttg catactctcc 180 atctttatcc aaaaatgtaaacttcctttg tcaatctatg gatatgccca tatatgaaag 240 agatgggtgg gtcaaaaagggatatcaaat gaagtgatag gggtcacaat ggggaaatgg 300 aagtggtaca taacattgccaaaataatgt gccactagaa atggtgtaaa ggctgtcttt 360 ttttttaaga aaagttattaccatgtattt tgtgaggcag gtttacaaca ctacaagtct 420 tgactaagaa ggaaagaggaaaaaagaaaa aacaccaata cccatattta aaaaaaaaaa 480 aatgatcata gtcttaggagttcatttaaa ccataggaac ttttcactta tctcatgtta 540 ggtgtaccag tcagtgattaagtagaacta caagttatat aggctgtatt gtttattgct 600 ggtttatgac cttaataaagtgtaatt atg tat tac cag cag ggt gtt ttt aac 654 Met Tyr Tyr Gln Gln GlyVal Phe Asn 1 5 tgt gac tat tgt ata aaa aca aat ctt gat atc cag aag cacatg aag 702 Cys Asp Tyr Cys Ile Lys Thr Asn Leu Asp Ile Gln Lys His MetLys 10 15 20 25 ttt gcg act ttc cac cct gcc cat ttt tgt aaa act gca gtcatc ttg 750 Phe Ala Thr Phe His Pro Ala His Phe Cys Lys Thr Ala Val IleLeu 30 35 40 gac ctt tta aac aca aat ttt aaa ctc aac caa gct gtg ata agcgga 798 Asp Leu Leu Asn Thr Asn Phe Lys Leu Asn Gln Ala Val Ile Ser Gly45 50 55 atg gtt act gtt tat act gtg gta tgt ttt tga ttacagcagataatgctttc 851 Met Val Thr Val Tyr Thr Val Val Cys Phe * 60 65ttttccagtc atctttgaga ataaaggaaa aaaaaaatct tcagatgcaa tggttttgtg 911tagcatcttg tctatcatgt tttgtaaatg ctggagaagc gtcgaccaat ttgacttaga 971gatggaatgt aactttgctt acaaaaattg ctattaaact cctacttaag gtgttctaat 1031tttctgtgag cacactaaaa acaaaaatat atgtgaataa aat 1074 <210> SEQ ID NO 36<211> LENGTH: 67 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 36 Met Tyr Tyr Gln Gln Gly Val Phe Asn Cys Asp Tyr Cys Ile LysThr 1 5 10 15 Asn Leu Asp Ile Gln Lys His Met Lys Phe Ala Thr Phe HisPro Ala 20 25 30 His Phe Cys Lys Thr Ala Val Ile Leu Asp Leu Leu Asn ThrAsn Phe 35 40 45 Lys Leu Asn Gln Ala Val Ile Ser Gly Met Val Thr Val TyrThr Val 50 55 60 Val Cys Phe 65 <210> SEQ ID NO 37 <211> LENGTH: 1356<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (553)...(1095) <400> SEQUENCE: 37ttctcccgca accttccctt cgctccctcc cgtccccccc agctcctagc ctccgactcc 60ctccccccct cacgcccgcc ctctcgcctt cgccgaacca aagtggatta attacacgct 120ttctgtttct ctccgtgctg ttctctcccg ctgtgcgcct gcccgcctct cgctgtcctc 180tctccccctc gccctctctt cggccccccc ctttcacgtt cactctgtct ctcccactat 240ctctgccccc ctctatcctt gatacaacag ctgacctcat ttcccgatac cttttccccc 300ccgaaaagta caacatctgg cccgccccag cccgaagaca gcccgtcctc cctggacaat 360cagacgaatt ctcccccccc ccccaaaaaa aaaagccatc cccccgctct gccccgtcgc 420acattcggcc cccgcgactc ggccagagcg gcgctggcag aggagtgtcc ggcaggaggg 480ccaacgcccg ctgttcggtt tgcgacacgc agcagggagg tgggcggcag cgtcgccggc 540ttccagacac ca atg gga atc cca atg ggg aag tcg atg ctg gtg ctt ctc 591Met Gly Ile Pro Met Gly Lys Ser Met Leu Val Leu Leu 1 5 10 acc ttc ttggcc ttc gcc tcg tgc tgc att gct gct tac cgc ccc agt 639 Thr Phe Leu AlaPhe Ala Ser Cys Cys Ile Ala Ala Tyr Arg Pro Ser 15 20 25 gag acc ctg tgcggc ggg gag ctg gtg gac acc ctc cag ttc gtc tgt 687 Glu Thr Leu Cys GlyGly Glu Leu Val Asp Thr Leu Gln Phe Val Cys 30 35 40 45 ggg gac cgc ggcttc tac ttc agc agg ccc gca agc cgt gtg agc cgt 735 Gly Asp Arg Gly PheTyr Phe Ser Arg Pro Ala Ser Arg Val Ser Arg 50 55 60 cgc agc cgt ggc atcgtt gag gag tgc tgt ttc cgc agc tgt gac ctg 783 Arg Ser Arg Gly Ile ValGlu Glu Cys Cys Phe Arg Ser Cys Asp Leu 65 70 75 gcc ctc ctg gag acg tactgt gct acc ccc gcc aag tcc gag agg gac 831 Ala Leu Leu Glu Thr Tyr CysAla Thr Pro Ala Lys Ser Glu Arg Asp 80 85 90 gtg tcg acc cct ccg acc gtgctt ccg gac aac ttc ccc aga tac ccc 879 Val Ser Thr Pro Pro Thr Val LeuPro Asp Asn Phe Pro Arg Tyr Pro 95 100 105 gtg ggc aag ttc ttc caa tatgac acc tgg aag cag tcc acc cag cgc 927 Val Gly Lys Phe Phe Gln Tyr AspThr Trp Lys Gln Ser Thr Gln Arg 110 115 120 125 ctg cgc agg ggc ctg cctgcc ctc ctg cgt gcc cgc cgg ggt cac gtg 975 Leu Arg Arg Gly Leu Pro AlaLeu Leu Arg Ala Arg Arg Gly His Val 130 135 140 ctc gcc aag gag ctc gaggcg ttc agg gag gcc aaa cgt cac cgt ccc 1023 Leu Ala Lys Glu Leu Glu AlaPhe Arg Glu Ala Lys Arg His Arg Pro 145 150 155 ctg att gct cta ccc acccaa gac ccc gcc cac ggg ggc gcc ccc cca 1071 Leu Ile Ala Leu Pro Thr GlnAsp Pro Ala His Gly Gly Ala Pro Pro 160 165 170 gag atg gcc agc aat cggaag tga gcaaaactgc cgcaagtctg cagcccggcg 1125 Glu Met Ala Ser Asn ArgLys * 175 180 ccaccatcct gcagcctcct cctgaccacg gacgtttcca tcaggttccatcccgaaaat 1185 ctctcggttc cacgtccccc tggggcttct cctgacccag tccccgtgccccgcctcccc 1245 gaaacaggct actctcctcg gccccctcca tcgggctgag gaagcacagcagcatcttca 1305 aacatgtaca aaatcgattg gctttaaaca cccttcacat accctccccc c1356 <210> SEQ ID NO 38 <211> LENGTH: 180 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <400> SEQUENCE: 38 Met Gly Ile Pro Met Gly LysSer Met Leu Val Leu Leu Thr Phe Leu 1 5 10 15 Ala Phe Ala Ser Cys CysIle Ala Ala Tyr Arg Pro Ser Glu Thr Leu 20 25 30 Cys Gly Gly Glu Leu ValAsp Thr Leu Gln Phe Val Cys Gly Asp Arg 35 40 45 Gly Phe Tyr Phe Ser ArgPro Ala Ser Arg Val Ser Arg Arg Ser Arg 50 55 60 Gly Ile Val Glu Glu CysCys Phe Arg Ser Cys Asp Leu Ala Leu Leu 65 70 75 80 Glu Thr Tyr Cys AlaThr Pro Ala Lys Ser Glu Arg Asp Val Ser Thr 85 90 95 Pro Pro Thr Val LeuPro Asp Asn Phe Pro Arg Tyr Pro Val Gly Lys 100 105 110 Phe Phe Gln TyrAsp Thr Trp Lys Gln Ser Thr Gln Arg Leu Arg Arg 115 120 125 Gly Leu ProAla Leu Leu Arg Ala Arg Arg Gly His Val Leu Ala Lys 130 135 140 Glu LeuGlu Ala Phe Arg Glu Ala Lys Arg His Arg Pro Leu Ile Ala 145 150 155 160Leu Pro Thr Gln Asp Pro Ala His Gly Gly Ala Pro Pro Glu Met Ala 165 170175 Ser Asn Arg Lys 180

That which is claimed is:
 1. A method for reducing growth of a cancerouscell comprising: contacting a cancerous cell with an amount of an agenteffective to redue tyrosine threonine kinase (TTK) polypeptide activityin the cell; wherein reduction of TTK polypeptide activity in thecancerous cell reduces growth of the cell.
 2. The method of claim 1wherein said reduction of TTK activity is a result of a reduction of TTKpolypeptide levels.
 3. The method of claim 2 wherein the agent is a TTKantisense polynucleotide.
 4. The method of claim 3 wherein the TTKantisense polynucleotide is contained in a viral-based vector.
 5. Themethod of claim 1 wherein said reduction of TTK activity is a result ofa reduction of TTK polynucleotide levels.
 6. The method of claim 1wherein the agent is a monoclonal antibody that specifically binds TTK.7. The method of claim 1 wherein the TTK polypeptide comprises the aminoacid sequence of SEQ ID NO:14.
 8. An assay for identifying a candidateagent that reduces growth of a cancerous cell, comprising: detecting theactivity of a TTK polypeptide in the presence of a candidate agent; andcomparing the activity of the TTK polypeptide in the presence of thecandidate agent relative to TTK polypeptide activity in the absence ofthe candidate agent; wherein a reduction of TTK activity in the presenceof the candidate agent relative to TTK activity in the absence of thecandidate agent indicates the candidate agent reduces growth of acancerous cell.
 9. The assay of claim 8, wherein said detecting steputilizes the polypeptide of SEQ ID NO:26 as a substrate.
 10. The assayof claim 8, wherein said detecting step uses a fragment of SEQ ID NO:26susceptible to TTK phosphorylation as a substrate.
 11. The assay ofclaim 10, wherein said fragment comprises the polypeptide of SEQ IDNO:27 or
 28. 12. The assay of claim 10 wherein the polypeptide fragmentis biotinylated.
 13. The assay of claim 8 wherein the TTK polypeptide isa product of expression using a system selected from the group ofbaculovirus, bacteria, yeast and mammalian systems.
 14. The assay ofclaim 13 wherein the TTK polypeptide is a product of expression using abaculovirus system.
 15. The method of claim 8 wherein the TTKpolypeptide comprises the amino acid sequence of SEQ ID NO:14.
 16. Amethod of identifying an agent that reduces TTK activity, the methodcomprising: contacting a cancerous cell displaying elevated expressionof a TTK-encoding polynucleotide with a candidate agent; and determiningthe effect of the candidate agent on TTK polypeptide activity; wherein adecrease in TTK activity indicates that the agent reduces TTK activityand inhibits growth of the cancerous cell.
 17. The method of claim 16wherein said reduction of TTK activity is a result of a reduction of TTKpolypeptide levels.
 18. The method of claim 16 wherein said reduction ofTTK activity is a result of a reduction of TTK mRNA levels.
 19. Themethod of claim 17 wherein the candidate agent is a TTK antisensepolynucleotide.
 20. The method of claim 19, wherein the TTK antisensepolynucleotide is contained in a viral-based vector.
 21. The method ofclaim 16 wherein the cancerous cell is a breast cancer cell.
 22. Themethod of claim 16 wherein the cancerous cell is a colon cancer cell.23. The method of claim 16 wherein TTK polypeptide comprises the aminoacid sequence of SEQ ID NO:14.
 24. The method according to claim 18,wherein TTK activity is detected by detecting expression of aTTK-encoding polynucleotide.
 25. A method of detecting cancer other thanovarian cancer in a subject, the method comprising: detecting a level ofexpression of a TTK polypeptide in a test cell obtained from a subjectsuspected of having cancer; and comparing the level of expression of theTTK polypeptide in the test cell to a level of expression in a normalnon-cancer cell of the same tissue type; wherein detection of anexpression level of TTK polypeptide in the test cell that issignificantly increased relative to the level of expression in thenormal non-cancer cell indicates that the subject has cancer other thanovarian cancer.
 26. The method of claim 25, wherein the test cell is acolon cell.
 27. The method of claim 25, wherein the test cell is abreast cell.
 28. A method of detecting cancer other than ovarian cancerin a subject, the method comprising: detecting a level of expression ofa TTK polynucleotide in a test cell obtained from a subject suspected ofhaving cancer; and comparing the level of expression of the TTKpolynucleotide in the test cell to a level of expression in a normalnon-cancer cell of the same tissue type; wherein detection of anexpression level of TTK polynucleotide in the test cell that issignificantly increased relative to the level of expression in thenormal non-cancer cell indicates that the subject has cancer other thanovarian cancer.
 29. The method of claim 29, wherein the test cell is acolon cell.
 30. The method of claim 29, wherein the test cell is abreast cell.
 31. A method for assessing the prognosis of a cancerousdisease other than ovarian cancer in a subject, the method comprising:detecting expression of a TTK-encoding polynucleotide in a test cancercell of a subject; and comparing a level of expression of a TTK-encodingpolynucleotide in the test cancer cell with a level of expression thepolynucleotide in a control non-cancer cell; wherein the level ofexpression of TTK in the test cancer cell relative to the level ofexpression in the control non-cancer cell is indicative of the prognosisof the cancerous disease
 32. The method of claim 31, wherein saiddetecting expression is by detection of a TTK-encoding transcript. 33.The method of claim 31, wherein the test cell is a colon cell.
 34. Themethod of claim 31, wherein the test cell is a breast cell.